Systems and methods for performing reactions in an unsealed environment

ABSTRACT

Open systems for performing submicroliter reactions are provided. The systems can include a support for performing the reaction; a liquid dispensing system for dispensing a submicroliter amount of a liquid to a site on or in the support; a temperature controlling device for regulating the temperature of the support; and an interface for controlling the amount of liquid dispensed from the liquid dispensing system are provided. Methods using the systems are also provided.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.09/266,409 to Thomas Becker, Hubert Köster and Charles Cantor, filedMar. 10, 1999, now U.S. Pat. No. 6,225,061, entitled “SYSTEMS ANDMETHODS FOR PERFORMING REACTIONS IN AN UNSEALED ENVIRONMENT”. Thesubject matter of this application is incorporated by reference in itsentirety.

FIELD OF THE INVENTION

The invention relates generally to systems and methods for performing areaction in a small volume without incurring an undesirable loss of thereaction volume due to evaporation, and more specifically to systems andmethods for performing reactions involving polymers, particularlybiopolymers, in a reaction volume of a few microliters or less in anunsealed environment.

BACKGROUND INFORMATION

Technological advances have allowed an examination of previouslyundiscernible phenomena. Such advances are particularly notable in thebiological sciences, where the chemical and physical structures of manybiopolymers have been described, and where such biopolymers, includingDNA and proteins, routinely are synthesized and sequenced.

Although methods such as nucleic acid sequencing and synthesis havecontributed to understanding the structure and function of biologicalmolecules and their relationships to disease, the limitations of suchmethods are apparent. For example, it generally is agreed that knowledgeof the entire sequence of the human genome would provide valuableinsight into the prevention and treatment of disease. The human genome,however, contains over one billion nucleotides and a huge expenditure oflabor and money would be required to sequence the entire human genome.Furthermore, using currently available methods, many years will berequired to see the project to completion.

Similarly, it is a goal of most clinical researchers to develop rapidand simple tests for determining whether an individual has a disease orpredisposition to a disease. In many cases, the signs and symptoms ofmany genetic diseases do not become apparent until an individual reachesa certain age or stage of development. Knowledge that an individual hasa predisposition to a genetic disease can allow the clinician to takeprophylactic measures to minimize or delay onset of the disease.Ideally, all individuals would be screened for potential geneticallydetermined diseases, including screening a large number of genes in eachindividual. Unfortunately, such routine screening currently is notfeasible because the assays are time consuming and the reagents forperforming such assays are expensive and limited in availability.

In an effort to reduce the time and cost for analyzing biopolymers,including genes and proteins, processes are being developed to automatethe analytic procedures. Automation provides a means for performingrepetitive processes almost continually, except for periodic breaks forequipment maintenance, and allows researchers and technical staff todevote more time to other endeavors, including interpreting the resultsproduced by the automated assays and trouble shooting problems that mayarise. Automation of repetitive processes also provides the advantagethat the likelihood of errors occurring, for example, due to fatigue ordistraction is reduced and, therefore, more accurate results can beobtained.

The application of nanotechnology to the biological sciences promises toprovide the next breakthroughs relating, for example, to the analysis,synthesis and utilization of biopolymers. Nanotechnology, which providesprocesses and apparatuses for performing procedures on a very smallscale, has been developed by the semiconductor industry in order toproduce smaller and smaller microchips, and to allow placement of acontinually increasing number of instructions on a microchip.

Efforts are in progress to apply nanotechnology to chemical andbiological procedures, thereby providing a means to perform assays invery small volumes, generally a few hundred nanoliters or less.Application of nanotechnology to biological assays can be particularlyvaluable because a critical limitation of many biological assays is theamount of biological material available for analysis. By performing suchassays in nanoliter volumes or smaller, the effective concentration of abiopolymer in a biological reaction is increased, thereby providing thenecessary kinetics for a biological reaction to proceed. In addition,the ability to perform biological assays in nanoliter volumes canprovide a significant cost savings because much smaller amounts ofreagents, which can be very expensive, can be utilized in the reactions.

The application of nanotechnology to biological assays has beenhindered, in part, by the difficulty in manipulating and maintainingsuch small volumes. Many biological assays, for example, are performedin aqueous conditions, using water as a solvent, and at elevatedtemperatures, generally at least 37° C., which is human bodytemperature. Water, like many liquid solvents, is susceptible toevaporation and, therefore, as the time or temperature of a reactionincreases, the loss of water due to evaporation increases and the volumeof the reaction decreases. As a result of evaporation, the effectiveconcentration of reagents in the reaction increases, thereby changingthe conditions of the reaction. Since most biological assays are quitesensitive to reaction conditions, loss of water or other solvent from areaction can result in an assay that produces spurious results. Any lossof a solvent such as water is particularly deleterious when the reactioncontains only a few hundred nanoliters or less of the liquid, since thereaction quickly can evaporate to dryness.

Various methods have been used to minimize the loss of solvent in areaction due to evaporation in biochemical assays. For example, reactionmixtures can be drawn into glass capillary tubes, which then are sealedat both ends for the reaction. Small volume glass capillary tubes can beexpensive, and the use of such tubes requires additional steps,including sealing and unsealing the tube, the latter which can produceglass shards.

In many cases, reaction mixtures are performed in a microcentrifuge tubeor other open chamber, and evaporation is minimized by overlaying thereaction mixture with wax, mineral oil, or other nonvolatile compoundduring the reaction. Such a method, again, requires additional steps,including removing the sealing material following the reaction. In orderto remove all or most of the sealing material, which can otherwisecontaminate the sample and hinder further analysis, some loss of thesample being assayed inevitably occurs. Since most biological samplesare limited to begin with, any loss of sample can preclude aninterpretation of the results of the assay. In general, any additionalmanipulations of a sample will incur extra cost, either in terms of timeor money, and loss or contamination of the sample.

More recently, biological reactions have been performed on microchips,which conveniently can be adapted to automated processes. Suchmicrochips have been designed having a system including, for example,chambers, which hold the reactants, and channels, which connect thechambers and in which the reactants can be mixed and a reactionperformed. Since the channels, in which the reaction occurs, provide asealed or closed environment, there is little or no evaporative loss ofthe reaction volume. Thus far, however, the technology for preparingsuch a device allows for the placement of only one or few of such closedsystems on a single microchip and, therefore, the number of reactionsthat can be performed at one time on a single chip is limited. Thus, aneed exists for systems useful for performing reactions in a volume of afew microliters or less in an unsealed environment. Therefore it is anobject here to provide systems and methods that satisfy this need andalso provide additional advantages.

SUMMARY OF THE INVENTION

Systems are provided for performing a reaction in an unsealedenvironment. The systems are used for any desired reaction, including,but not limited to in situ biopolymer or polymer synthesis, such asnucleic acid and protein syntheses, protein and nucleic acid sequencingmethods, such as oligonucleotide-based primer extension, nucleic acidamplification reactions, protein and nucleic acid protease- ornuclease-based degradations and others.

A system as disclosed herein is an open system for performing areaction, such as a synthetic reaction or an assay, particularly in asubmicroliter volume. The systems can include a support for performingthe reaction; a nanoliter dispensing pipette for dispensing asubmicroliter amount of a liquid to a target site on the support; atemperature controlling device for regulating the temperature of thesurface of the support; and means for controlling the amount of liquiddispensed, where the amount of liquid dispensed corresponds to theamount of liquid evaporated from the support. A means for controllingthe amount of liquid dispensed can include computer software thatcalculates the rate of evaporation and signals the dispensing pipette todeliver an amount of the liquid that corresponds to the amount lost dueto evaporation. A means for controlling the amount of liquid dispensedalso can be manual input, which can be performed by an individual.

A system as disclosed herein also can include a temperature measuringdevice for measuring the temperature of the surface of the support. Thesupport can be any support having a surface, including, for example, abead, pin, comb, wafer, well or microchip, and the support can befunctionalized such that a substrate, for example, a biopolymer can belinked, either directly or indirectly via covalent or non-covalentinteractions, to the support and immobilized.

An open system, as disclosed herein, also can include a solid support,which has a target site that can contain a volume of liquid, forexample, a reaction mixture; a liquid dispensing system, which candispense a liquid to the target site; a temperature controlling system,which can regulate the temperature of the solid support; and aninterface, which can indicate an amount of liquid to be dispensed to thetarget site from the liquid dispensing system. An interface can include,for example, a computer using an appropriate algorithm. A computer canmonitor the temperature of the solid support and, based on variousparameters, including, for example, the chemical nature of the liquid,the surface area of the liquid exposed to the environment, and the timethe liquid is maintained at a particular temperature, and can provideinformation as to the amount of liquid to be dispensed from the liquiddispensing system to the target site to maintain the liquid at apredetermined volume. Based on that information, the liquid dispensingsystem can be manipulated manually, to dispense the liquid to the targetsite, or can be controlled automatically, for example, by interfacing itwith the computer. In a system as disclosed herein, the amount of liquiddispensed from a liquid dispensing system to a target site generallycorresponds to an amount of liquid lost from the target site due toevaporation, although the amount added also can be an initial amountadded to a target site or an amount added to modify the conditions of areaction.

An open system, as disclosed herein, also can include a solid supporthaving a target site; a liquid dispensing system, which can dispense aliquid to the target site; a temperature controlling system, whichregulates the temperature of the solid support; and means for regulatingan amount of liquid dispensed from the liquid dispensing system. Inaddition, an open system, as disclosed herein, can have a means forcontaining a reaction mixture; a means for dispensing a liquid; a meansfor controlling the temperature of the reaction volume containing means;and means for regulating an amount of liquid dispensed from the liquiddispensing means.

A means for regulating an amount of liquid dispensed can be a computerhaving an appropriate algorithm. Such a computer can interface with thesolid support, thereby monitoring the temperature of the support, andcan provide an indication of an amount of liquid to be dispensed to atarget site to maintain a predetermined volume, for example, of areaction volume. The computer can cause to be displayed the amount ofliquid to be dispensed, such that an individual can manipulate theliquid dispensing system and dispense the liquid, or the computer canfurther interface with the liquid dispensing system, thereby causing theamount of liquid to be dispensed. In addition, charts can be developedthat predict the amount and rate of evaporation of a particular solventat a particular temperature and, based on such charts, an individual canmanipulate the liquid dispensing system as necessary. Also, a decreasein the volume of a liquid due to evaporation can be identified directlyby including the liquid in a circuit, wherein, when the liquid fallsbelow a predetermined point, the circuit is broken, thereby indicatingthat a liquid should be dispensed to the target site until the circuitis reestablished.

Methods for maintaining a volume of a liquid in an unsealed environmentalso are provided. A method for performing a reaction in a predeterminedsubmicroliter volume in the open can be performed by dispensing thepredetermined submicroliter volume of liquid onto the surface of asupport; optionally monitoring the temperature of the substrate;determining the amount or rate of evaporation of the liquid from thesupport; and dispensing a further amount of the liquid to the surface ofthe support, wherein the further amount dispensed corresponds to theamount lost from the support due to evaporation, thereby maintaining thereaction volume at a predetermined volume throughout the course of thereaction. Such a method also can be performed, for example, bydetermining the temperature of a solid support, which has a target sitethat can contain a volume of liquid; and, based on the temperature,dispensing at the target site an amount of liquid required to maintain apredetermined volume of the liquid. The amount of liquid to be dispensedcan be determined using a computer algorithm, which, based on variousparameters, including the temperature of the support, the chemicalnature of the liquid, the surface area of the liquid exposed to theenvironment, and the volume to be maintained, can indicate the amount ofliquid that evaporates from the site and, therefore, the amount ofliquid to be dispensed to maintain a predetermined volume. The volume ofa liquid on a target site also can be monitored, for example, bymicroscopic examination, using an appropriate optical system or a videoimaging device, such that, as the volume of a liquid at a target sitedecreases due to evaporation, a corresponding amount of liquid can bedispensed to maintain the volume within acceptable parameters.

Methods for performing a reaction in an unsealed environment also areprovided. Such a method can be performed, for example, by determiningthe temperature of a solid support, which has a target site containing avolume of the reaction mixture, or determining the rate or amount ofevaporation of liquid from the reaction mixture; and dispensing into thereaction mixture an amount of liquid required to maintain the volume ata predetermined level. Such a method is particularly useful where thereaction mixture has a volume of a few microliters or less, particularlya volume of about 500 nanoliters or less. The disclosed methods also areuseful for performing submicroliter reactions at temperatures where thevapor pressure of a liquid in the reaction mixture is undesirably high,for example, about 2.5 kiloPascals (kPa) or greater, particularly about5 kPa or greater, or about 10 kPa or greater, such that evaporation ofthe liquid can substantially change the volume of the reaction mixtureand adversely affect the reaction. As such, the disclosed methods areuseful for performing various chemical, physical and biologicalreactions, for example, a polymerase chain reaction, or a nucleic acidor polypeptide synthesis or sequencing reaction or other reaction orassay performed on a solid support.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 provides an exemplary embodiment of an open system for performinga reaction in an unsealed environment. A liquid is contained on a targetsite on the surface of a microchip, which is in a holder that isintegrated with a Peltier element and thermistor. The temperature of thePeltier element/thermistor is regulated by a temperature controller,which is interfaced with a computer, and the temperature of the chip ismonitored. The computer, which also is interfaced with the nanoliterdispensing device, receives input of the measured temperature,calculates the amount of liquid that evaporates from the target site,and signals the dispensing device to dispense an amount of liquid thatcorresponds to the amount that evaporates, thereby maintaining thevolume of liquid on the target site at a predetermined level.

DETAILED DESCRIPTION OF THE INVENTION

A system is provided for performing reactions in an unsealedenvironment, including reactions performed in submicroliter volumes. Anopen system as disclosed herein solves the previously intractableproblems caused by evaporation of a liquid solvent during a reaction,including, for example, the concomitant increase in the effective saltconcentration, which can inhibit a reaction or lead to spurious results.An additional advantage of the disclosed open systems and methods ofperforming a reaction in an unsealed environment is that fewer steps arerequired to perform a reaction because the reaction volume need not besealed or covered with a protective layer such as mineral oil to preventevaporation, such manipulations further requiring that the volume laterbe unsealed or isolated from the protective layer. As a result, thelikelihood that any sample will be lost due to the additionalmanipulations is reduced.

An open system as disclosed herein provides the further advantage that“single tube” reactions can be performed, wherein a number of differentreactions are performed at the same target site. The ability to performsingle tube reactions further reduces the likelihood that any samplewill be lost due to transferring a material from one tube to another forperforming different reactions, and facilitates the automation ofchemical and biological reactions. It should be recognized that suchreactions, while referred to as “single tube” reactions, need notliterally be performed in a “tube,” but can be performed at any targetsite having the characteristics disclosed herein.

The disclosed open systems are useful for performing liquid handling,for example, for performing reactions such as polymerase chain reaction(PCR), DNA sequencing and enzymatic digestion reactions. Such reactionscan be performed directly on the surface of a modified silicon chip thatcan be used for mass spectrometric detection of the resulting products,or allows on-line monitoring of fluorescent or luminescent signals. Itis important to prevent evaporation of a solvent, generally water, of areaction to prevent alterations in concentrations of reactants or othercomponents such as salts during the reactions. Typically, evaporation isprevented by performing reactions in sealed or closed environments. Themethods and systems disclosed herein permit reactions to be performed,for example, directly on the surface of a microchip without a need for alid or sealing. This achieved by replacing nanoliter amounts of water orwater/glycerol or other reaction mixture components in the reactionmixture using drop-on-demand systems, which compensate for loss ofsolvent by evaporation.

An open system as disclosed herein can include a support for performingthe reaction; a nanoliter dispensing pipette for dispensing asubmicroliter amount of a liquid onto the surface of the support; atemperature controlling device for regulating the temperature of atarget site on the support, particularly of a liquid at the target site;and means for controlling the amount of liquid dispensed, wherein theamount of liquid dispensed corresponds to the amount of evaporation of athe liquid from the support. A system as disclosed herein also caninclude a temperature measuring device for measuring the temperature ofthe surface of the support. The support can be any support having asurface, including, for example, a bead, pin, comb, wafer, well ormicrochip, and the support can be functionalized such that a biopolymercan be linked to the support and immobilized.

A means for controlling the amount of liquid dispensed can includecomputer software that calculates the rate or amount of evaporation ofthe liquid and signals the dispensing pipette to deliver an amount ofthe liquid that corresponds to the amount lost due to evaporation. Ameans for controlling the amount of liquid dispensed also can be manualinput, which can be performed by an individual. In addition, a means forcontrolling the amount of liquid dispensed can be a system thatdetermines when a meniscus of a liquid decreases below a predeterminedpoint. Such a system can be, for example, an electrical circuit, whichis broken when the meniscus falls below a predetermined point; or aphotometric or spectrophotometric system, which detects a change indiffraction, transmission or absorbance of photons when the meniscusfalls below a predetermined point. Such a meniscus determining meansconveniently can provide an interface between the target site and theliquid dispensing system. A means for controlling the amount of liquiddispensed also can be a system for determining the conductivity (orresistivity) of the liquid, which changes in parallel with a change inthe reaction volume, such that, when the conductivity (or resistivity)reaches a predetermined value, an indication is provided as to an amountof liquid to be dispensed to the target site to the maintain the liquidat a predetermined volume.

An open system as disclosed allows a reaction to be performed in anunsealed environment. An open system can include a solid support, whichhas a target site that contains the reaction mixture; a liquiddispensing system; a temperature controlling system, which regulates thetemperature of the solid support; and an interface that regulates anamount of liquid dispensed from the liquid dispensing system. Theinterface can indicate an amount of liquid to be dispensed based, forexample, on the temperature of the solid support or the decrease of ameniscus below a predetermined point, and the amount of liquid dispensedcan correlate with the amount of liquid that evaporates from a reactionmixture on the solid support. Also provided is a system having means fordispensing a liquid; means for containing a reaction volume; means forcontrolling the temperature of the reaction volume containing means; andmeans for regulating an amount of liquid dispensed from the liquiddispensing means based on the temperature of the reaction volumecontaining means. A means for containing a reaction volume can be asolid support having, for example, a well or pin, or a barrier, whichcan be a physical or chemical barrier.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which this invention belongs. All patents, pending and publishedapplications and publications referred to herein are incorporated byreference.

As used herein, the term “unsealed environment,” when used in referenceto a volume of a liquid, means that there is no particular barrierpresent to prevent substantial evaporation of the liquid into theenvironment. For purposes herein, substantial evaporation occurs whenevaporation occurs at a rate or amount that alters the reactionconditions before the reaction of interest is completed. This isparticularly problematic for reactions that are performed in wells or onthe surface of solids small volumes, typically submicroliter volumes.Hence, methods and systems are provided herein to permit such reactionsto be performed.

As used herein, an open system refers to the systems disclosed hereinfor maintaining a volume of a liquid in an unsealed environment. Thesecan be referred to generally as “open systems” although such systems canbe sealed from the air, such as under inert gas or in a box or othercontainer. As noted an open system is one in which evaporation occursduring the reaction of interest in an amount or rate such thatconditions of the reaction are altered, such by a change inconcentration of critical components, such as salt concentrations. Thiswill occur if small volume, such as submicroliter reactions, areperformed in wells or on the surface of a solid support. The disclosedsystems and methods, thus, are distinguishable from systems and methodsfor performing a reaction, for example, in an unsealed microcapillarytube or in a channel on a microchip because, even though a liquid may bein direct contact to the open air in such systems, the amount ofevaporation that occurs is not unacceptable either because theevaporation is desired, or because the surface area of the liquid incontact with the gaseous medium is so small with respect to the volumeof the liquid that any evaporation that occurs during the reactionperiod does not deleteriously affect or alter the reaction conditions.

As used herein, the term “liquid dispensing system” means a device thatcan transfer a predetermined amount of liquid to a target site. Theamount of liquid dispensed and the rate at which the liquid dispensingsystem dispenses the liquid to a target site, which can contain areaction mixture, can be adjusted manually or automatically, therebyallowing a predetermined volume of the liquid to be maintained at thetarget site.

As used herein, the term “liquid” is used broadly to mean a non-solid,non-gaseous material, which can be homogeneous or heterogeneous and cancontain one or more solid or gaseous materials dissolved or suspendedtherein. In general, a liquid is a component of a reaction mixture thatis susceptible to evaporation under the conditions of the reaction. Inparticular, the liquid can be a solvent, in which a reaction isperformed, for example water or glycerol/water or buffer or reactionmixture, where the reaction is performed in an aqueous solution. Theliquid can be any non-solid, non-gaseous solvent or other component of areaction mixture that is susceptible to evaporative loss, for example,acetonitrile, which can be a solvent for a nucleic acid synthesisreaction; formamide, which can be a liquid component of a nucleic acidhybridization reaction; piperidine, which is a liquid component of anucleic acid sequencing reaction; or any other non-aqueous solvent orother liquid component. A liquid can contain dissolved or suspendedcomponents, which can be useful, for example, for initiating,terminating or changing the conditions of a reaction, therebyfacilitating the performance of single tube reactions.

As used herein, the term “reaction mixture” refers to any solution inwhich a chemical, physical or biological change is effected. In general,a change to a molecule is effected, although changes to cells also arecontemplated. A reaction mixture can contain a solvent, which provides,in part, appropriate conditions for the change to be effected, and asubstrate, upon which the change is effected. A reaction mixture alsocan contain various reagents, including buffers, salts, and metalcofactors, and can contain reagents specific to a reaction, for example,enzymes, nucleoside triphosphates, amino acids, and the like. Forconvenience, reference is made herein generally to a “component” of areaction, wherein the component can be a cell or molecule present in areaction mixture, including, for example, a biopolymer or a productthereof.

As used herein, the term “biopolymer” is used to mean a biologicalmolecule composed of two or more monomeric subunits, or derivativesthereof, which are linked by a bond or a macromolecule. A biopolymer canbe, for example, a polynucleotide, a polypeptide, a carbohydrate, or alipid, or derivatives or combinations thereof, for example, a nucleicacid molecule containing a peptide nucleic acid portion or aglycoprotein, respectively. The methods and systems herein, thoughdescribed with reference to biopolymers, can be adapted for use withother synthetic schemes and assays, such as organic syntheses ofpharmaceuticals, or inorganics and any other reaction or assay performedon a solid support or in a well in nanoliter volumes.

As used herein, a biological particle refers to a virus, such as a viralvector or viral capsid without or without packaged nucleic acid, phage,including a phage vector or phage capsid, with or without encapsulatednucleotide acid, a single cell, including eukaryotic and prokaryoticcells or fragments thereof, and other such biological materials.

As used herein, the term “polynucleotide” refers to an oligomer orpolymer containing at least two linked nucleotides or nucleotidederivatives, including a deoxyribonucleic acid (DNA), a ribonucleic acid(RNA), and a DNA or RNA derivative containing, for example, a nucleotideanalog or a “backbone” bond other than a phosphodiester bond, forexample, a phosphotriester bond, a phosphoramidate bond, aphophorothioate bond, a thioester bond, or a peptide bond (peptidenucleic acid). The term “oligonucleotide” also is used hereinessentially synonymously with “polynucleotide,” although those in theart will recognize that oligonucleotides, for example, PCR primers,generally are less than about fifty to one hundred nucleotides inlength.

Nucleotide analogs contained in a polynucleotide can be, for example,mass modified nucleotides, which allows for mass differentiation ofpolynucleotides; nucleotides containing a detectable label such as afluorescent, radioactive, luminescent or chemiluminescent label, whichallows for detection of a polynucleotide; or nucleotides containing areactive group such as biotin or a thiol group, which facilitatesimmobilization of a polynucleotide to a solid support. A polynucleotidealso can contain one or more backbone bonds that are selectivelycleavable, for example, chemically, enzymatically or photolytically. Forexample, a polynucleotide can include one or more deoxyribonucleotides,followed by one or more ribonucleotides, which can be followed by one ormore deoxyribonucleotides, such a sequence being cleavable at theribonucleotide sequence by base hydrolysis. A polynucleotide also cancontain one or more bonds that are relatively resistant to cleavage, forexample, a chimeric oligonucleotide primer, which can includenucleotides linked by peptide nucleic acid bonds and at least onenucleotide at the 3′ end, which is linked by a phosphodiester bond, orthe like, and is capable of being extended by a polymerase. Peptidenucleic acid sequences can be prepared using well known methods (see,for example, Weiler et al., Nucleic acids Res. 25:2792-2799 (1997)).

A polynucleotide can be a portion of a larger nucleic acid molecule, forexample, a portion of a gene, which can contain a polymorphic region, ora portion of an extragenic region of a chromosome, for example, aportion of a region of nucleotide repeats such as a short tandem repeat(STR) locus, a variable number of tandem repeats (VNTR) locus, amicrosatellite locus or a minisatellite locus. A polynucleotide also canbe single stranded or double stranded, including, for example, a DNA-RNAhybrid, or can be triple stranded or four stranded. Where thepolynucleotide is double stranded DNA, it can be in an A, B, L or Zconfiguration, and a single polynucleotide can contain combinations ofsuch configurations.

As used herein, the term “polypeptide,” means at least two amino acids,or amino acid derivatives, including mass modified amino acids and aminoacid analogs, that are linked by a peptide bond, which can be a modifiedpeptide bond. A polypeptide can be translated from a polynucleotide,which can include at least a portion of a coding sequence, or a portionof a nucleotide sequence that is not naturally translated due, forexample, to it being located in a reading frame other than a codingframe, or it being an intron sequence, a 3′ or 5′ untranslated sequence,a regulatory sequence such as a promoter, or the like. A polypeptidealso can be chemically synthesized and can be modified by chemical orenzymatic methods following translation or chemical synthesis. The terms“polypeptide,” “peptide” and “protein” are used essentially synonymouslyherein, although the skilled artisan will recognize that peptidesgenerally contain fewer than about fifty to one hundred amino acidresidues, and that proteins often are obtained from a natural source andcan contain, for example, post-translational modifications. Apolypeptide can be post-translationally modified by phosphorylation(phosphoproteins), glycosylation (glycoproteins, proteoglycans), and thelike, which can be performed in a cell or in a reaction in vitro.

As used herein, a reaction mixture used in an open system or method asdisclosed herein can have any volume from a few picoliters or less tohundreds of liters or more. An open system or method as disclosed hereinis particularly useful where a volume to be maintained is critical inorder for a reaction to occur, and where the volume to be maintained isnot amenable to simple inspection or measurement. As such, the disclosedsystems and methods generally are useful where the reaction volume isabout 500 milliliters or less; are more useful where the reaction volumeis about 5 milliliters or less; are most useful where the reactionvolume is in the “submilliliter” range, for example, about 500microliters, or about 50 microliters or about 5 microliters or less; andare particularly useful where the reaction volume is a “submicroliter”reaction volume, which can be measured in nanoliters, for example, about500 nanoliters or less, or 50 nanoliters or less or 10 nanoliters orless, or can be measured in picoliters, for example, about 500picoliters or less or about 50 picoliters or less. For convenience ofdiscussion, the term “submicroliter” is used herein to refer to areaction volume less than about one microliter, although it will bereadily apparent to those in the art that the systems and methodsdisclosed herein are applicable to subnanoliter reaction volumes aswell. A reaction mixture is contained in or on a target site on a solidsupport.

As used herein, the term “solid support” means a non-gaseous, non-liquidmaterial having a surface. Thus, a solid support can be a flat surfaceconstructed, for example, of glass, silicon, metal, plastic or acomposite; or can be in the form of a bead such as a silica gel, acontrolled pore glass, a magnetic or cellulose bead; or can be a pin,including an array of pins suitable for combinatorial synthesis oranalysis.

As used herein, the term “target site” refers to a specific locus on asolid support that can contain a liquid. A solid support contains one ormore target sites, which can be arranged randomly or in ordered array orother pattern. In particular, a target site restricts growth of a liquidto the “z” direction of an xyz coordinate. Thus, a target site can be,for example, a well or pit, a pin or bead, or a physical barrier that ispositioned on a surface of the solid support, or combinations thereofsuch as a beads on a chip, chips in wells, or the like. A target sitecan be physically placed onto the support, can be etched on a surface ofthe support, can be a “tower” that remains following etching around alocus, or can be defined by physico-chemical parameters such as relativehydrophilicity, hydrophobicity, or any other surface chemistry thatallows a liquid to grow primarily in the z direction. A solid supportcan have a single target site, or can contain a number of target sites,which can be the same or different, and where the solid support containsmore than one target site, the target sites can be arranged in anypattern, including, for example, an array, in which the location of eachtarget site is defined.

As used herein, the term “predetermined volume” is used to mean anydesired volume of a liquid. For example, where it is desirable toperform a reaction in a 5 microliter volume, 5 microliters is thepredetermined volume. Similarly, where it is desired to deposit 200nanoliters at a target site, 200 nanoliters is the predetermined volume.

As used herein, the term “maintain a volume of a liquid” refers to apredetermined volume of the liquid and means that the volume of theliquid is kept within an acceptable amount of the predetermined volume.An acceptable amount of a predetermined volume is an amount that iswithin 80% or more of the predetermined volume, generally within about90% or more, and particularly about 95%, or about 98% or more of thepredetermined volume.

As use herein, a volume of a liquid at a target site is maintainedwithin a predetermined volume by dispensing an amount of liquid to thetarget site. In one embodiment, the amount of liquid dispensed to atarget site is based on the evaporation rate of the liquid from thetarget site, such that the amount of liquid dispensed corresponds to theamount of liquid lost from the volume due to evaporation. As usedherein, the term “corresponds,” when used in reference to the amount ofliquid being added to the reaction mixture (“amount added”) and theamount of liquid that evaporates from the reaction mixture (“amountlost”), means that the amount added is, within an acceptable margin oferror, equal to the amount lost. An acceptable margin of error is suchthat the amount added is within 20% or less of the amount lost,generally within about 10% or less, and particularly about 5% or less,or about 2% or less of the amount lost. An acceptable margin of errorcan be determined based, for example, on the susceptibility of areaction to the effective concentration of one or more reactants in thereaction. In another embodiment, the disclosed systems and methods allowa predetermined amount of a liquid to be dispensed to a target site, forexample, to initiate a reaction, to dilute a reaction, or to change theconditions of a reaction.

As used herein, the term “temperature controlling system” means a devicefor regulating the temperature of a solid support, particularly thetemperature of a liquid present at a target site on a surface of thesolid support. A temperature controlling system useful in an open systemas disclosed can increase the temperature of a solid support,particularly a target site on the support, or decrease the temperatureof the support, as desired. Temperature controlling systems are wellknown and readily available to those in the art, and are selected, inpart, based on the range of temperatures desired, the physicalcharacteristics of the solid support, and its facility of incorporationinto a system as disclosed. A temperature controlling system can be, forexample, an electrically or electromagnetically regulated heatingelement or heating/cooling element, such as a Peltier element, or asystem that allows contacting the support with, for example, dry ice,liquid nitrogen, or a bath or stream of water maintained at a desiredtemperature.

As used herein, the term “interface” refers to a system forcommunicating an amount of liquid to be dispensed to a target site tomaintain a predetermined volume. As such, an interface provides a meansfor controlling an amount of liquid dispensed from a liquid dispensingsystem. An interface can be in communication, either directly orindirectly, with the target site, with the liquid dispensing system, orwith both.

As used herein, the abbreviations for amino acids and protective groupsand other such abbreviations are in accord with their common usage and,if appropriate, the IUPAC-IUB Commission on Biochemical Nomenclature[see, (1972) Biochem. 11: 1726].

Systems

Systems are provided for performing a reaction in an unsealedenvironment. The disclosed systems and methods provide a means ofmaintaining a volume of a liquid, for example, a reaction mixture,present in an unsealed environment and, therefore, susceptible to lossof volume by evaporation. In general, the environment into whichevaporation can occur is a volume of a gaseous medium, which can be, butneed not be, substantially greater than the volume of liquid. Arelatively large surface of the liquid can be in direct contact with theenvironment, and a substantial amount of a liquid can evaporate into theenvironment, for example, ten percent or more of the total volume, suchthat a substantial change in the effective concentration of reactantswould occur if the amount of liquid lost due to evaporation is notreplaced by a corresponding amount of the liquid dispensed to thevolume.

In a system or method as disclosed herein, the liquid generally ispresent on a surface of a solid support, at a target site, and theenvironment into which evaporation can occur is air. Many liquids andreactants, including biopolymers, suitable for use in a disclosed systemor method are susceptible, for example, to oxidation. Accordingly, anopen system as disclosed herein can be placed in a vessel in which theenvironment can be controlled, for example, the environment can begaseous medium such as nitrogen, an inert gas such as argon, or othergaseous medium. It should be recognized, therefore, that an unsealedenvironment may be isolated from the “open air,” but nevertheless can beconsidered an “unsealed environment” for purposes of the presentdisclosure provided that a liquid, the volume of which is to bemaintained, is in contact with a gaseous medium, into which the liquidcan evaporate. Further in this regard, various reactions must beperformed under conditions of low pressure or high pressure, where therate of evaporation of a liquid is greater than or less than,respectively, the rate of evaporation of the liquid in open air. An opensystem as disclosed herein also can be used for performing suchreactions, as well as reactions that contain evaporation suppressants oragents that alter the freezing point or boiling point of the liquid,particularly such agents that do not affect a reaction, or forperforming reactions in the light, which can be any spectrum of light,or in the dark.

The disclosed systems and methods provide a means to maintain a volumeof a liquid at a predetermined volume, where the volume otherwise woulddecrease below the predetermined volume due to evaporation. An opensystem can include a solid support having a target site, which cancontain a volume of liquid; a liquid dispensing system, which candispense a liquid to the target site; a temperature controlling system,which can regulate the temperature of the solid support; and aninterface, which can indicate an amount of liquid to be dispensed fromthe liquid dispensing system. In an open system as disclosed herein, theamount of liquid dispensed from a liquid dispensing system cancorrespond to an amount of liquid lost by evaporation, or can be anypredetermined amount of liquid.

The liquid present on a target site can be, for example, a solvent orother component of a reaction mixture. Other components of a reactionmixture can include a substrate, for example, a cell, a biopolymer or anorganic or inorganic molecule, and the volume of the reaction can be anydesired volume, particularly a submicroliter volume. An open system asdisclosed herein can be particularly useful for synthesizing biopolymerssuch as polynucleotides, polypeptides, polysaccharides and the like,including for synthesizing combinatorial libraries of molecules such asbiopolymers, and for performing biological reactions, or chemicalreactions using a biopolymer as a substrate, in submicroliter volumes,without concern that evaporation of a liquid from the reaction mixturewill undesirably affect the reaction, and, additionally, allows forperforming such reactions as single tube reactions.

An open system also can contain a solid support having a target site; aliquid dispensing system, which can dispense a liquid to the targetsite; a temperature controlling system, which regulates the temperatureof the solid support; and means for regulating an amount of liquiddispensed from the liquid dispensing system. Any means for regulating anamount of liquid dispensed from the liquid dispensing system can beused, including manual manipulation of the liquid dispensing system byan individual monitoring the system, or automatic control of the liquiddispensing system due to an interface between the liquid dispensingsystem and the temperature controlling device or a temperature sensingdevice in contact with the solid support.

Interface

An interface generally is a component of an automated or semi-automatedopen system for maintaining a volume of a liquid at a target site. Inparticular, an interface can be a computerized system that receivesinput relevant to the volume of a liquid at a target site and, based onthat input, provides an instruction to the liquid dispensing system todispense an amount of liquid that corresponds to an amount of liquidlost from the target site. Thus, an interface for regulating an amountof liquid dispensed by a liquid dispensing system can be, for example, asystem for detecting the level of a meniscus, or a computer forreceiving input of data from which the volume or level can becalculated. Thus, the interface can include a computer programmed withan appropriate algorithm or software for calculating such level.

Input relevant to the volume of a liquid at a target site can beobtained directly, for example, by detecting a decrease in the level ofa meniscus of the liquid or the level of a drop of the liquid below apredetermined point. As disclosed herein, a liquid on a target site canbe, for example, in a well or cylinder. In such a case, wherein theliquid is physically surrounded by a barrier, a meniscus forms in theliquid. In addition, a liquid can be placed as a drop on the targetsite, wherein the liquid is constrained, for example, by thephysico-chemical characteristics of the target site. In either case, thelevel of the liquid can be monitored by detecting a decrease in thelevel of the meniscus or the drop of liquid.

A decrease in a meniscus below a predetermined point can be detected,for example, by including the liquid in a circuit. In such a system,when the meniscus falls below a predetermined point, which is the pointrequired for the circuit to be complete, a change in the circuit isdetected. The interface, upon receiving such input, can indicate that avolume of liquid is to be dispensed to the target site by the liquiddispensing system, until the circuit is reestablished, at which timedispensing of the liquid is terminated. Such a circuit conveniently canbe constructed into a microchip using well known methods ofphotolithography and microelectronics.

Similarly, where the liquid on the target site has a meniscus or is inthe form of a droplet, such input can be obtained by detecting a changein the diffraction, transmission or absorbance of photons as the volumeof liquid decreases below a level defined by the positions of anappropriate light source and detector. A system using fiber optics canbe useful for monitoring the level of a liquid on a target site and,conveniently, can be included in a detection system, if desired, tomonitor the extent of a reaction. As well as the direct methodsexemplified above, input relevant to the volume of a liquid at a targetsite also can be obtained indirectly, for example, using an algorithmthat determines the rate of evaporation from the target site based onthe temperature of the support containing the target site, the time thetemperature has been maintained, and the vapor pressure of the liquid.

A computer with appropriate inputs and outputs, for example, can be usedto monitor the temperature of the solid support and, based on variousparameters, including, for example, the chemical nature of the liquid,the surface area of the liquid exposed to the environment, and the timethe liquid is maintained at a particular temperature, can estimate therate of evaporation of a liquid from the target site, and, through aninterface, communicate an amount of liquid to be dispensed from theliquid dispensing system to the target site to maintain the liquid at apredetermined volume. For example, a means for regulating an amount ofliquid dispensed to a target site can interface with a liquid dispensingsystem such as a nanoliter dispensing system to compensate forevaporation, thereby maintaining the volume of a liquid at a target siteat a predetermined volume (see, e.g., FIG. 1). A computer can directlycontrol the liquid dispensing system to dispense a desired volume, whichcorresponds to the amount of liquid that evaporates from the targetsite. The amount of evaporation will depend, in part, on the temperatureof the target site, which can be on the surface of a microchip presentin a holder that is integrated with, for example, a Peltier element, anda thermistor. The temperature of the support and, therefore, the liquidat a target site, can be any temperature, which can be adjusted based oninput from the computer, which is interfaced with the temperaturecontrolling system. Based on the temperature of the microchip, thecomputer can calculate a rate or amount of evaporation and signal thenanoliter dispensing system accordingly.

A temperature sensing device such as a thermistor produces a signal thatindicates the temperature of the support, for example, a microchipsupport (see FIG. 1). The support temperature signal can be provided tothe computer, directly or through the temperature controlling system.Based on the temperature of the support, programming in the computerdetermines the amount or rate of evaporation and, therefore, a volume ofliquid that can be dispensed to the target site to maintain the volumeof the liquid at a predetermined level. The computer can provide thetemperature controlling system with a signal that indicates thetemperature to which the support will be set. Upon receiving thetemperature setting signal, the temperature controlling system producesa Peltier element control signal, which directs the Peltier element toadjust the support to the indicated temperature.

An interface need not be directly connected to or control the liquiddispensing system, but can be connected instead to a display, whichindicates the amount of liquid needed to be dispensed to maintain thevolume of the liquid at a predetermined volume. An individual then canmanipulate the liquid dispensing system. In addition, the interface neednot be directly connected to the solid support, but can be connectedinstead to the temperature controlling system and, based on the settingof the temperature controlling system, the chemical and physical natureof the solid support, and the time the temperature is applied to thesupport, can determine the temperature of the solid support and,therefore, the amount of liquid to be dispensed to the target site. Theinterface then can display the amount of liquid to be dispensed suchthat an individual can manipulate the liquid dispensing system, or cantransmit the information to the liquid dispensing system, therebyautomatically controlling the system.

An open system, as disclosed herein, also can contain means forcontaining a volume of a liquid; means for dispensing a liquid; meansfor controlling the temperature of the reaction volume containing means;and means for regulating an amount of liquid dispensed from the liquiddispensing means. A means for regulating an amount of liquid dispensedcan include an interface, for example, a computer programmed withsoftware for calculating the appropriate rate or amount. A computer caninterface, for example, with the solid support, thereby monitoring thetemperature of the support, and can indicate an amount of liquid to bedispensed to a target site to maintain a predetermined volume of theliquid. The computer can display the amount of liquid to be dispensedand an operator can manipulate the liquid dispensing system such thatthe amount of liquid is dispensed, or the computer can further interfacewith the liquid dispensing system, thereby causing the amount of liquidto be dispensed.

Solid Supports

A solid support useful in an open system for maintaining a volume of aliquid at a predetermined volume can be constructed of any materialhaving a surface, which can be flat or geometrically altered, forexample, to include wells. The solid support is any known to those ofskill in the art as matrix for performing synthetic reactions andassays. It can be fabricated from silicon, glass, silicon-coatedmaterials, metal, a composite, a polymeric material such as a plastic, apolymer-grafted material, such as a metal-grafted polymer, or othermaterial as disclosed herein. This material can be furtherfunctionalized, as necessary, for example, chemically, to enhance orpermit linkage of molecules or other particles, such as cells or cellmembranes or viral envelopes or other such biological materials, ofinterest. The surface of a support can be modified, such as by radiationgrafting of a suitable polymer on the surface and derivatization thereofto render it suitable for binding capturing a molecule or particle, suchas a cell. The support may also include beads linked thereto (see,copending allowed U.S. application Ser. No. 08/746,036, copending U.S.application Ser. No. 08/933,792, and International application No.PCT/US97/20194, which claims priority to the U.S. applications). It mayalso include dendrite trees of captured material, or combinations ofsuch additional components. A solid support can have one or more targetsites, each of which can contain or retain a volume of a liquid.

By way of example, a solid support can be a flat surface such as a glassfiber filter, a glass surface, a silicon or silicon dioxide surface, acomposite surface, or a metal surface, including a steel, gold, silver,aluminum or copper surface, a plastic material, including polyethylene,polypropylene, polyamide or polyvinylidenedifluoride, which further canbe in the form of multiwell plate or a membrane; can be in the form of abead (or other geometry) or particle, such as a silica gel, a controlledpore glass, a magnetic or cellulose bead, which can be in a pit of aflat surface such as a wafer, for example, a silicon wafer; or can be apin, including an array of pins suitable for combinatorial synthesis oranalysis (see, e.g., International PCT application No. WO98/20019),comb, microchip. The skilled artisan will recognize that variousfactors, including the size and shape of the support and the chemicaland physical stability of the support to the conditions to which it willbe exposed, will be considered in selecting a particular solid supportfor use in a disclosed system or method.

A solid support contains one or more target sites, which can contain avolume of a liquid. A target site can be, for example, a well, pit,channel, or other depression, with or without rims, on the surface of asolid support; can be a pin, bead or other material, which can bepositioned on a surface of a solid support; or can be a physical barriersuch as a cylinder, cone or other such barrier positioned on a surfaceof a solid support.

A target site also can be, for example, a reservoir or reaction chamber,which is attached to a solid support (see, for example, Walters et al.,Anal. Chem. 70:5172-5176 (1998). In addition, a target site can beetched, for example, on a surface of a silicon wafer using aphotolithographic method (see, for example, Woolley et al. (Anal. Chem.68:4081-4086 (1996)). Photolithography allows the construction of verysmall target sites, including wells or towers, and, for example, hasbeen used in combination with wet chemical-etching to construct“picoliter vials” on microchips (Clark et al. CHEMTECH 28:20-25 (1998)).

A support also can be a glass or silicon surface containing wells havinga very thin base that is transparent to electromagnetic radiation of adesired wavelength, such as laser light, thereby permitting measurementof parameters, such as volume, or an excitation wavelength forfluorescence measurement.

A target site also can be defined by physico-chemical parameters such ashydrophilicity, hydrophobicity, the presence of acidic or basic groups,groups capable of forming a salt bridge, or any surface chemistry thatallows a liquid to grow primarily in the z direction. For example, wherethe liquid to be placed on a target site is water or an aqueoussolution, the target site can be defined by a hydrophilic areasurrounded by a hydrophobic area on the surface of a solid support, orby a series of rows, alternately having less hydrophobic rows and morehydrophobic rows, whereby the aqueous mixture is constrained to the lesshydrophobic rows. With respect to such a target site, the aqueoussolution is dispensed, for example, onto the hydrophilic area, and isconstrained from spreading from the target site due to the adjacent andsurrounding hydrophobic area. Conversely, where the liquid is a nonpolarliquid, it is dispensed onto a hydrophobic region and is constrained inthat region due to an adjacent hydrophilic region or a region or that isless hydrophobic that the region to which the liquid is applied.

A solid support can have a single target site, or can contain a numberof target sites, for example, 2 sites, 10 sites, 16 sites, 100 sites,144 sites, 384 sites, 1000 sites, or more, all or some of which can bethe same or can be different. Where a solid support contains more thanone target site and, therefore, can contain, for example, more than onereaction mixture, the characteristics that define each target site servenot only to constrain a reaction mixture, but also to preventintermingling of different reaction mixtures or other liquids on thesupport. In addition, where a solid support contains more than onetarget site, the target sites can be arranged in any pattern, forexample, in a line, a spiral, concentric circles, rows, or an array ofrows and columns. Furthermore, the location of each target site of anumber of target sites on a support can be defined. The availability ofsuch addressable target sites on a solid support allows multiplereactions to be performed in parallel and is convenient, for example,for performing multiplex reactions, for including control reactions withtest reactions such that all are performed under identical conditions,for performing a similar reaction under different conditions, or forperforming different reactions.

Immobilization of a Reagent to a Solid Support

A substrate or other component of a reaction mixture can be immobilizedto a solid support, particularly to a target site on the support, by acovalent interaction or a noncovalent interaction that is stable to theparticular conditions of the reaction, as desired. A biopolymer, forexample, can be immobilized directly to a solid support, or indirectly,for example, by immobilization to a spacer molecule, which isimmobilized to the support. Furthermore, a spacer molecule can be a partof the biopolymer to be immobilized, or can be a separate molecule thatdirectly or indirectly binds the biopolymer, for example, anoligonucleotide including a spacer nucleotide sequence and asufficiently complementary probe or primer sequence, which can hybridizeto a polynucleotide biopolymer.

Immobilization of a biopolymer can be mediated by a specific bindingreaction, for example, by hybridization of a first nucleic acid moleculeto a sufficiently complementary second nucleic acid, one of which isimmobilized to the support. Similarly, immobilization can be through afirst protein to a second protein, one of which is immobilized to thesupport, for example, an antibody and a polypeptide, which can beexpressed on the surface of a cell, having an epitope recognized by theantibody; or an enzyme and its substrate; or any pair of proteinscapable of homodimer or heterodimer formation. In addition,immobilization can be between a nucleic acid binding protein and apolynucleotide containing the sequence recognized by the bindingprotein.

A crosslinking agent also can be used to immobilize a substrate or othercomponent of a reaction mixture to a solid support, through a reversibleor irreversible linkage. A useful crosslinking agent can be any agent,including a homo-bifunctional or hetero-bifunctional agent, that iscapable of reacting with a functional group present on a surface of theinsoluble support and with a functional group present in the substrateor other component to be immobilized to the support. Useful bifunctionalcross-linking agents include N-succinimidyl (4-iodacetyl) aminobenzoate(SIAB), dimaleimide, dithio-bis-nitrobenzoic acid (DTNB),N-succinimidyl-S-acetyl-thioacetate (SATA),N-succinimidyl-3-(2-pyridyidithiol propionate (SPDP), succinimidyl4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) and6-hydrazinonicotimide (HYNIC) (see, also, Wong “Chemistry of ProteinConjugation and Cross-Linking,” (CRC Press 1991); Hermanson,“Bioconjugate Techniques” (Academic Press 1995)).

Immobilization of a substrate or other component of a reaction to asolid support can be particularly useful where the crosslink is mediatedby a selectively cleavable linker, which can be cleaved under definedconditions. A biopolymer, for example, can be directly linked to a solidsupport via a reversible or irreversible bond between an appropriatefunctionality (L′) on the biopolymer and an appropriate functionality(L) on the solid support, or on a molecule linked thereto, for example,a spacer molecule. Selectively cleavable linkers include photocleavablelinkers and chemically cleavable linkers (see, e.g., International PCTapplication No. WO98/20019), and enzymatically cleavable linkers such asa polynucleotide sequence containing a particular restrictionendonuclease site or a RNase digestion site or a polypeptide sequencecontaining a particular peptidase site.

Photocleavable linkers, which are cleaved upon exposure to light(Goldmacher et al., Bioconj. Chem. 3:104-107 (1992)), include anitrobenzyl group as a photocleavable protective group for cysteine(Hazum et al., in Pept. Proc. Eur. Pept. Symp., 16th (ed. K. Brunfeldt,1981), pages 105-110); water soluble photocleavable copolymers,including hydroxypropylmethacrylamide copolymer, glycine copolymer,fluorescein copolymer and methylrhodamine copolymer (Yen et al.,Makromol. Chem 190:69-82 (1989)); a cross-linker and reagent thatundergoes photolytic degradation upon exposure to near UV light (350 nm;Goldmacher et al., Bioconj. Chem. 3:104-107 (1992);nitrobenzyloxycarbonyl chloride cross-linking agents (Senter et al.,Photochem. Photobiol 42:231-237 (1985); and 3-amino-(2-nitrophenyl)propionic acid (Brown et al., Molecular Diversity, pages 4-12 (1995);Rothschild et al., Nucl. Acids Res. 24:361-66 (1996)). A photocleavablebond such as a charge transfer complex or a labile bond formed betweenrelatively stable organic radicals can be useful, for example, where thesample is to be examined by mass spectrometry.

A linkage also can be formed with L′ being a quaternary ammonium group.Where the sample is to be examined by mass spectrometry, the surface ofthe solid support also can carry a negative charge, which can repel, forexample, a negatively charged nucleic acid backbone and facilitatedesorption of a polynucleotide to be detected. Desorption can occureither by the heat created by the laser pulse or, depending on L′, byspecific absorption of laser energy, which is in resonance with the L′chromophore.

The L-L′ chemistry can be a type of disulfide bond, which is chemicallycleavable using mercaptoethanol or dithioerythrol; a biotin/streptavidinsystem; a heterobifunctional derivative of a trityl ether group that canbe cleaved under mildly acidic conditions as well as under conditions ofmass spectrometry (Köster et al., Tetrahed. Lett. 31:7095 (1990)); alevulinyl group cleavable under almost neutral conditions with ahydrazinium/acetate buffer; an arginine-arginine or lysine-lysine bond,which is cleavable by an endopeptidase enzyme such as trypsin; apyrophosphate bond, which is cleavable by a pyrophosphatase; or aribonucleotide bond in an oligodeoxynucleotide sequence, which can becleaved by a ribonuclease or alkali. In addition to acid-labile trityllinkers, acid cleavable linkers include bis-maleimideothoxypropane;adipic acid dihydrazide linkers (Fattom et al., Infect. Immun.60:584-589 (1992)); and acid labile transferrin conjugates that containa sufficient portion of transferrin to permit entry into theintracellular transferrin cycling pathway (see, for example, Welhöner etal., J. Biol. Chem. 266:4309-4314 (1991)).

The L and L′ functionalities also can form a charge transfer complex andthereby form a temporary L-L′ linkage; a charge-transfer “band” can bedetermined by UV/vis spectrometry (R. Foster, “Organic Charge TransferComplexes” (Academic Press 1 969)), and the laser energy can be tuned tothe corresponding energy of the charge-transfer wavelength so as toeffect specific desorption of a sample for a solid support. A reversibleL-L′ linkage also can be generated by homolytically forming relativelystable radicals, which, under the influence of a laser pulse, forexample, during mass spectrometry, desorption and ionization occurs atthe radical position. Those skilled in the art will recognize that otherorganic radicals can be selected and that, in relation to thedissociation energies needed to homolytically cleave the bond betweenthem, a corresponding laser wavelength can be selected (C. Wentrup,“Reactive Molecules” (John Wiley & Sons 1984)).

Thiol-reactive functional groups are particularly useful forimmobilizing a biopolymer to a solid support. Thiol-reactive functionalgroups react with a nucleophilic thiol moiety to produce a covalentbond, for example, a disulfide bond or thioether bond. In general, thiolgroups are good nucleophiles, and preferred thiol-reactive functionalgroups are reactive electrophiles. Thiol-reactive functional groups areknown in the art and include, for example, haloacetyls such asiodoacetyl; diazoketones; epoxy ketones; α,β-unsaturated carbonyls suchas α,β-enones; and other reactive Michael acceptors, includingmaleimide, acid halides, benzyl halides, and the like.

A free thiol group present on a polypeptide or incorporated into apolynucleotide can react with a free thiol group such as aniodoacetyl-modified surface (or other thiol-reactive surfacefunctionality) of the support through disulfide bond formation, therebyimmobilizing the biopolymer to the support. In addition to beingreversible, for example, by exposing the bond to reducing conditions,thiol reactive linkages also provide the advantage that reaction of athiol group can be prevented temporarily by blocking with an appropriateprotecting group (Greene and Wuts, in “Protective Groups in OrganicSynthesis” 2nd ed. (John Wiley & Sons 1991)).

A polynucleotide can be modified at the 3′-terminus or 5′-terminus byreaction with a disulfide-containing modifying a reagent, or byenzymatically or non-enzymatically attaching a thiolated primer. A5′-phosphoramidate functionality can also provide an attachment pointfor a thiol or disulfide-containing cytosine or deoxycytosine residue. Adisulfide-modified nucleic acid can be reduced in a reaction using, forexample, tris-(2-carboxyethyl)phosphine (TCEP), at a concentration ofabout 1 mM to 100 mM, preferably about 10 mM; a pH of about 3 to 6,preferably about pH 4.5; a temperature in the range of 20° C. to 45° C.,preferably about 37° C.; and for a time period in the range of about 1hour to 10 hours, preferably about 5 hours; or using dithiothreitol in aconcentration of about 25 mM to 100 mM, depending on whether thereactant is isolated; at a pH in the range of 6 to 10, preferably aboutpH 8; at a temperature of about 25° C. to 45° C., preferably about 37°C.; and for a time of about 1 hour to 10 hours, preferably about 5hours. Use of TCE provides an advantage in the low pH at which it isreactive, which effectively protonates thiols, thereby suppressingnucleophilic reactions of thiols and resulting in fewer side reactionsthan with other disulfide reducing agents used at higher pH ranges.

Temperature Controlling System

The temperature of a solid support having a target site is maintainedusing a temperature controlling system. An open system for maintaining avolume of a liquid at a predetermined volume can include a temperaturecontrolling system, which can heat or cool a solid support, particularlya target site on the support, to a desired temperature. A temperaturecontrolling system is selected, in part, based on the purpose for whichthe open system is to used, for example, the reactions to be performedusing the open system. As such, a temperature controlling system can beselected that can cool a solid support, for example, to a temperature of4° C. or less, or 0° C. or less, or −20° C. or less, or −80° C. or less,including, if desired, to about the temperature of dry ice, or liquidnitrogen, or liquid helium; or can heat a solid support, for example, to25° C. or more, or 37° C. or more, or 45° C. or more, or 65° C. or more,or 90° C. or more, including, if desired, to temperatures greater than100° C. A temperature controlling system that can heat or cool a solidsupport within a temperature range of about −20° C. to about 95° C. canbe particularly useful as a component of an open system that is to beused for performing biological reactions or reactions involvingbiological materials, since the reaction volume, including the reactantscan be maintained at a low temperature prior to initiating the reaction,then can be adjusted to the appropriate temperature or temperatures forperforming the desired reaction.

A thermoelectric module can be particularly useful as a temperaturecontrolling system in an open system as disclosed herein. Athermoelectric module is a solid state device that can be used as a heatpump, utilizing the Peltier effect, to move heat. Depending on thedirection that module moves heat, it can be used to heat or cool a solidsupport. A single thermoelectric module generally can achieve atemperature difference of about 60° C. to 70° C., and several suchmodules can be used in combination to attain a temperature difference ofup to 131° C. Furthermore, by reversing the direction of the current tothe module, the direction heat is moved can be reversed. Thus, thethermoelectric module can be used to reversibly heat and cool a supportand, therefore, a reaction mixture such a s a PCR reaction located at atarget site on the support. Thermoelectric modules are commerciallyavailable (Melcor, Trenton N.J.; Americool, Nashua N.H.).

A temperature controlling system such as the Peltier Thermal Cycler(PTC−200 DNA Engine; M.J. Research, Inc., Watertown Mass.) is an exampleof a temperature controlling system that can be integrated into an opensystem as disclosed herein. The PTC−200 DNA Engine utilizes aPeltier-Joule heat pump; has a temperature range of −5° C. to 105° C.;provides temperature homogeneity of samples within 30 seconds ofreaching 90° C.; accepts a variety of sample supports, includingmicroscope slides and multi-well plates; and conveniently can beinterfaced with a computer. An LFI-3526 system, which contains a 22WPeltier element controlled by a programmable thermoelectric temperaturecontroller (Wavelength Electronic, Inc., Bozeman Mont.; see Example 1),is another example of a temperature controlling system useful in an opensystem as disclosed.

An electronic temperature controlling system also can be incorporatedinto an open system as disclosed (see Burns et al., Proc. Natl. Acad.Sci., USA 93:5556-5561 (1996)). An electronic temperature controllingsystem can be conveniently constructed, for example, in a microchipusing well known methods (see, for example, Woolley et al. Anal. Chem.68:4081-4086 (1996)). Such an electronic temperature controlling systemallows thermal cycling using a pulsed width modifier and, therefore, canbe useful, for example, for performing PCR reactions (Woolley etal.(Anal. Chem. 68:4081-4086 (1996); see, also, Ross et al., Anal. Chem.70:2067-2073 (1998); Belgrader et al., Clin. Chem. 44:2191-2194 (1998)).

A temperature controlling system also can include a temperaturemeasuring system, which can be used to determine the temperature of asolid support, particularly of a liquid present at a target site on thesurface of the support. A temperature measuring system can be, forexample, a thermocouple, thermometer, or the like, which can be incontact with a liquid on the support and, therefore, directly determinethe temperature of the liquid, or can be in contact with the solidsupport, thereby providing an indication of the temperature of theliquid. A temperature also can be, for example, an infrared detector,which can monitor the temperature of a liquid at a target site withoutcontacting the support. The use of thermocouples can be particularlyconvenient because they can be very small in size and can beconstructed, for example, into microchips (see Woolley et al. (Anal.Chem. 68:4081-4086 (1996)). A thermocouple can be in direct contact witha target site, including with a reaction volume at each target site inan array on a solid support, thereby allowing precise monitoring of thetemperature of each reaction simultaneously. Input from suchthermocouples can be incorporated into an algorithm that allows acalculation of the evaporation rate of liquid from each reaction mixturein an array, and, through the appropriate interface, indicates an amountof liquid to be dispensed to each target site that corresponds to theamount that evaporates from a reaction mixture. Such a means formonitoring the temperature of a number of reaction volumessimultaneously can be particularly useful since different reactions maybe being performed at different sites in the array, or because the rateof heating or cooling of different reaction mixtures on a solid supportare not identical due, for example, to inhomogeneities in the support orto different concentrations of reagents in a mixture.

The temperature generated by the temperature controlling system and,therefore, the temperature of the solid support, also can be determinedbased on the particular setting of the temperature controlling system,the physical nature of the solid support, and the time the temperatureis applied to the support. Such temperatures can be calculated based onknown parameters, or can be determined empirically by heating or coolinga support for incremental periods of time, at incremental temperatures,and measuring the temperature of the support accordingly.

Liquid Dispensing/Removing Systems Dispensing

A liquid dispensing system can be an active apparatus, which can be amechanical, electrical, pressure or pneumatic driven liquid dispensingsystem, for example, a piezo electric pipette driven by mechanicalpressure; or can be a passive apparatus, which contains a reservoir. Inaddition, a liquid dispensing system can contain a heating element, forexample, microresistors, which provides the ability to maintain a liquidin the system at a desired temperature, for example, at or near areaction temperature.

A liquid dispensing system can include a single fluid transmittingvesicle or multiple vesicles, which can be manipulated independently ortogether in parallel. A fluid transmitting vesicle can be a solidvesicle, to which the liquid can adsorb and be transferred, or can havea bore, through which the liquid is transferred. Thus, a fluidtransmitting vesicle can be a pipet, particularly a micropipet, whichcontains a chamber for holding or transferring the liquid and an endfrom which the liquid can be dispensed to a target site; a pin tool,which can have a bore, or can be solid vesicle, which, when dipped intoa chamber holding a liquid, adsorbs a volume of the liquid, which thencan be transferred to a target site; or a liquid sonicating, vaporizingor ink jet device, which contains a chamber for holding the liquid, andan end from which the liquid is dispensed in droplets, the volume andrate of dispensing of which can be adjusted as desired. A fluidtransmitting vesicle can be formed of a metal, composite, glass, silica,or polymeric material, or any other suitable material. A nanoliterliquid dispensing system such as a nanoliter pipet can be particularlyuseful in a system as disclosed. Nanoliter dispensing systems areprovided, for example, in copending allowed U.S. application Ser. No.08/787,639, U.S. application Ser. No. 08/786,988, and International PCTapplication No. WO 98/20166, which claims priority to the U.S.applications.

A liquid dispensing system can be part of a liquid handling system,which can contain, in addition to the liquid dispensing system, achamber for holding a liquid to be dispensed. Such a chamber can be usedto directly provide the liquid dispensing system with the appropriateliquid to be dispensed, or can be connected to the liquid dispensingsystem by a conduit, which mediates transfer of the liquid from theholding chamber to the dispensing system. A conduit can be any suitableconduit, for example, plastic or stainless steel tubing, and can beparticularly useful if it can be sterilized without impairing itsfunction. Where it is desirable to dispense a liquid to a target site ata particular temperature, the liquid dispensing system, as discussedabove, or a component of a liquid handling system can be maintained atthe particular temperature such that the liquid is dispensed at thedesired temperature. An advantage of a liquid handling system is that itcan contain more than one holding chamber and, therefore, canconveniently allow more than one liquid to be dispensed from a singleliquid dispensing system, for example, from a pin having a bore, withouta need to change the position of the liquid dispensing system withrespect to the target site. Such a system is particularly convenientwhere the fluid transmitting vesicle, for example, a pin tool, has narray of fluid transmitting vesicles, which are positioned with respectto a corresponding array of target sites on a solid support.

A liquid dispensing system allows an amount of liquid, preferably acontrolled amount, to be dispensed to a target site. The liquid can bedispensed as a continuous stream, or as droplets, which can be dispensedcontinuously or in a burst mode. The amount of liquid dispensed can beany amount, as desired, including a submicroliter amount or less, andcan be dispensed for the purpose of maintaining a liquid at apredetermined volume, or for initiating, terminating or changing theconditions of a reaction at the target site.

A liquid dispensing system can dispense one or more liquids to a singletarget site, or can dispense one or more liquids serially or in parallelto multiple target sites, which can be in an array. A liquid dispensingsystem useful for dispensing a predetermined amount of a liquid inparallel can include, for example, an assembly of liquid transmittingsystems such as pins, each of which can have a narrow interior chambersuitable for holding a volume of the liquid to be dispensed (see, forexample, International PCT application No. WO98/20166). The pins can befit inside a housing, which can have an interior chamber connected, forexample, to a pressure source that regulates the flow of liquid througha pin, thereby allowing controlled dispensing of a predetermined volumeof the liquid. Alternatively, the liquid dispensing system can include ajet assembly and a transducer element mounted to a pin, and can dispensean amount of liquid to a target site by spraying the liquid from thepin, or by allowing a drop of the liquid to form on the tip of the pin,where it can be contacted to the target site and dispensed.

A liquid dispensing system can include a single chamber for holding aliquid and, therefore, allow a single liquid to be dispensed, or cancontain several chambers, each of which can hold a different liquid andvariably can be in connection with the fluid transmitting vesicle. Assuch, a liquid dispensing system can include a selection element having,for example, a pressure source or a piezoelectric element coupled to aliquid holding chamber and in communication with the fluid dispensingvesicle such that, at a selected pressure condition or a selectedvoltage, a particular liquid is dispensed at a predetermined amount.Such a selection element conveniently can be interfaced with andcontrolled by a computer algorithm, which can be monitoring a rate ofevaporation of a liquid from a target site, and can allow one or moreliquids to be dispensed to a target site, or serially or in parallel toa plurality of target sites. In addition, a liquid dispensing system candispense a liquid at any desired temperature, particularly thetemperature at which a reaction is performed, or a temperature such asabout 4° C., which, for example, can suspend a biological reaction. Ananoliter dispensing device, such as the NANO-PLOTTER NP1c (sold byGeSim; Dresden Germany) is an example of a liquid dispensing system thatcan be incorporated into an open system as disclosed (see Examples 1 and2). The Nano-Plotter is a modular device that can be combined in avariety of ways depending upon the intended application. It is designedto spot microdroplets arrays onto flat substrates or microwell plates.The device as sold contains from one to eight micropipettes. For useherein, the device can be modified by including heating/cooling elementsor heating means to heat the reservoir or the micropipetter or otherportions thereof, preferably the surface of the target support, to heator cool the liquid or surface prior to dispensing liquid to avoid atemperature gradient or change upon addition of liquid to a reactionmixture. Other nanoliter dispensing devices can also be used or adaptedfor use in these systems (see, e.g., copending allowed U.S. applicationSer. No. 08/787,639, U.S. application Ser. No. 08/786,988, andInternational PCT application No. WO 98/20166, which claims priority tothe U.S. applications, which describe nanoliter dispensing devices andsystems).

The liquid dispensing system can dispense a liquid, which generally isreagent grade or better, or can dispense a solution containing theliquid. For example, in one aspect, the methods as disclosed providediagnostic assays, the results of which can be analyzed using, forexample, mass spectrometry, capillary electrophoresis, a charge coupleddevice, or a fiber optic system. Where a method such as MALDI-TOF massspectrometry is used to analyze a component of a reaction, the sample tobe analyzed is mixed with an appropriate matrix material (see, forexample, U.S. Pat. No. 5,605,798; International PCT application No.WO96/29431; International PCT application No. WO98/20019). As such, aliquid dispensing system can be used to dispense a matrix solution to atarget site, prior to subjecting the sample at the target site to massspectrometry.

Liquid Removing

An open system as disclosed herein also can include a device forremoving a liquid, which can be a reaction mixture, from a target siteand transferring it to another target site or to a chamber for disposal.Such a device provides a convenient means to terminate a reaction,change the reaction conditions, wash a sample, or the like. Accordingly,in an embodiment, the liquid dispensing system also can function toremove a liquid from a target site. The liquid dispensing system, orindependent device, can remove a liquid from a target site by contactingthe fluid transmitting vesicle to the liquid to be removed and, forexample, allowing capillary action to draw the liquid into the vesicleor applying a negative pressure to the vesicle. The removed liquid canbe transferred to another location, which can be another target site ora chamber for disposal, and the fluid transmitting vesicle can bewashed, if desired, and positioned for further use. A device forremoving a liquid from a target site also can be a device thatfacilitates evaporation of the liquid from the target site, for example,a fan or other device for passing a stream of air or other gas over theliquid.

Regulation of Liquid Dispensing System

The liquid dispensing system is regulated so as to dispense a definedamount of a liquid to a target site. The amount of liquid dispensed cancorrespond to an amount of liquid lost due to evaporation or can be anydesired amount of liquid, including a reaction mixture or a solutioncontaining components of a reaction mixture. The liquid dispensingsystem can be regulated manually or can be regulated semi-automaticallyor automatically based, for example, on instructions from a computer orother signal transmitting system, which can be interfaced with thetemperature controlling system (or a temperature sensing device), withthe liquid dispensing system, or with the temperature controlling system(or temperature sensing device) and the liquid dispensing system.

A signal transmitting system can be any system that indicates an amountof liquid to be dispensed. For example, where the amount of liquid to bedispensed corresponds to an amount of liquid lost from a target site dueto evaporation, the signal transmitting system can be any system thatprovides an indication of the amount of liquid lost. The amount of aliquid lost from a volume in an unsealed environment depends on thevapor pressure of the liquid, which is a function, in part, of thetemperature; the surface area of the liquid exposed to the environment;the nature of the environment, including, for example, its relativehumidity; and the time during which liquid can be lost. Since theseparameters will be known or are determinable for a particular set ofconditions, tables can be constructed for predicting an amount of aparticular liquid that will be lost in a period of time from a knownvolume of the liquid applied to a particular target site at a knowntemperature. Accordingly, for purposes of the present disclosure, such atable is considered a signal transmitting system because an individual,monitoring the temperature and time of a particular reaction, canmanipulate the liquid dispensing system to dispense an amount of liquidas indicated by the table.

For semi-automatic or automatic regulation of the liquid dispensingsystem, the signal transmitting system can be a conventional digitaldata processing system, for example, an IBM PC compatible computersystem, an Apple computer or a UNIX based system, that is suitable forprocessing data and for executing program instructions that will provideinformation that can be communicated to the liquid dispensing system.Such a signal transmitting system can be any type of system suitable forprocessing a program of instructions that will operate the liquiddispensing system, although the system need not necessarily beprogrammable and can be a single board computer having a firmware memoryfor storing instructions relevant to regulating the liquid dispensingsystem.

A signal transmitting system can monitor the temperature of a solidsupport containing a target site directly, for example, by interfacingwith a temperature sensing device, or indirectly, for example, based onthe setting of the temperature controlling device and informationrelating to the chemical and physical nature of the solid support.Alternatively, or in addition, the signal transmitting system can beinterfaced with the liquid dispensing system. For example, forsemi-automatic operation, the signal transmitting system can beinterfaced with either the liquid dispensing system or the temperaturecontrolling system, and the component of the system that is notinterfaced with the signal transmitting system can be operated manuallyby an individual. More conveniently, however, the temperaturecontrolling system and the liquid dispensing system are interfaced withthe signal transmitting system, and the entire system is operatedautomatically.

A signal transmitting system also can interface directly with the targetsite, including directly with the liquid. For example, a signaltransmitting system can include a circuit in a microchip, where thecircuit is interrupted by a well in the chip. Upon dispensing a liquidinto such a well, the liquid can complete the circuit. In particular,the volume of liquid required to complete the circuit is indicative ofthe predetermined volume, which is to be maintained. Accordingly, whereevaporation of the liquid occurs to the point that the level of theliquid decreases below the level required to maintain the circuit, anindication is provided that a liquid is to be dispensed to the targetsite. Upon dispensing a sufficient volume of the liquid such that thecircuit is reestablished, dispensing of the liquid is terminated.

A signal transmitting system also can include a microbalance, which candetect minute changes in the weight of a support due to evaporation of aliquid from the surface of the support. In addition, a signaltransmitting system can include a light source, which can be of anydesired wavelength, and a detector appropriate for the light source. Thelight can be provided to a target site, for example, using a fiberoptic, and the amount of diffraction of the light, or the transmissionor absorption of photons can be monitored. A change in the amount ofsuch a parameter can indicate, similarly to the circuit system discussedabove, that the level of the liquid has decreased below a predeterminedvalue, or, as discussed below, that an undesirable amount of evaporationhas occurred. Such information then can be communicated such that anamount of liquid is dispensed to the target site that corresponds to theamount that has evaporated, thereby maintaining the volume of the liquidat a predetermined level.

A spectrophotometric detector system, for example, can include a laser,which can be a helium-argon laser, a helium-neon laser, an ultravioletlaser, or a laser that emits green or blue light, or can be a lightemitting diode (LED), for example, a blue or a green LED. Such adetector system can be particularly useful where the support is a glassor silicon chip, which has wells or the like having a base that allowstransmission of the particular wavelength of light. Using such asupport, the light can be transmitted from below the chip, through thesample, and can be detected by a detector placed above the well. Thelight transmitting system and detector can be a single source, or can bearranged in an array that corresponds to positions of the target siteson the support. Furthermore, such a spectrophotometric system can beseparate from the open system for maintaining a volume of a liquid in anunsealed environment, and the support can be repositioned to thedetector system when desired. Preferably, however, thespectrophotometric system is integrated into the open system, thusallowing online monitoring of a reaction. In such an integrated system,a temperature controlling system such as a Peltier element isconstructed with holes at positions corresponding to the target sites,particularly to the position at which the light source transmits thelight to the support.

The use of a spectrophotometric system can allow monitoring of areaction, for example, where the reactants are labeled with anappropriate fluorescent, luminescent or chemiluminescent moiety, forexample, a reaction performed using the TaqMan™ assay (see Example 1).The light transmitting system and detector are selected based on theparticular wavelength of light desired. Such a spectrophotometric systemalso is useful for monitoring the volume of a liquid at a target site,as disclosed above, for example, by detecting a change in thediffraction, transmission or absorbance of photons that reach thedetector. Depending on the particular liquid at the target site,including the desired predetermined volume to be maintained, thereactants, when present, in the liquid, and the like, tables can beconstructed that indicate, for example, the amount of light transmittedto a detector that signals an undesirable amount of evaporation of theliquid, such that a liquid dispensing system dispenses an amount ofliquid that corresponds to the amount that evaporated.

Detection Systems

A reaction mixture, including a component added to the mixture, forexample, a substrate, or an intermediate or product produced by thereaction, can be monitored using any detection system appropriate forthe material being examined. The detection system is selected based inthe particular material to be detected, and can be matched with aparticular label where the material to be detected based on identifyingthe presence of a label attached to the material. Since the disclosedsystems and methods are particularly useful for performing reactions insmall volumes, particularly submicroliter volumes, the material to bedetected generally is present in only a very small amount. Accordingly,the detection system is selected, in part, on its sensitivity fordetecting the material.

A detection system can be a photometric or spectrophotometric system,which can detect ultraviolet, visible or infrared light, includingfluorescence or chemiluminescence; a radiation detection system; aspectroscopic system such as nuclear magnetic resonance spectroscopy,mass spectrometry or surface enhanced Raman spectrometry; a chargecoupled device; a system such as gel or capillary electrophoresis or gelexclusion chromatography; or other detection system known in the art, orcombinations thereof.

A mass spectrometry detection system can be useful in an open system asdisclosed because it can detect the presence of very small amounts of amaterial, for example, a biopolymer, and, at the same time provides anindication of the identity of the detected material. In addition, massspectrometry does not require labelling a material to be detected,although the materials can be “labeled,” for example, by incorporatingmass differentiating functional groups into the materials where amultiplex reaction is to be performed. Mass spectrometry also is usefulbecause the systems and methods disclosed herein can utilize a solidsupport such as a microchip, which can be introduced conveniently intothe mass spectrometer.

A useful mass spectrometry detection system can be any of variousformats, including ionization (I) techniques such as matrix assistedlaser desorption (MALDI), continuous or pulsed electrospray (ESI),ionspray, thermospray, or massive cluster impact (MCI). Such ion sourcescan be matched conveniently with a detection format, including linear orreflectron time-of-flight (TOF), single or multiple quadruple, single ormultiple magnetic sector, Fourier transform ion cyclotron resonance(FTICR), ion trap, and combinations thereof to yield a hybrid detector,for example, ion-trap/time-of-flight. For ionization, numerousmatrix/wavelength combinations (MALDI) or solvent combinations (ESI) canbe employed. MALDI-TOF mass spectrometry, including delayed extractionMALDI-TOF mass spectrometry is particularly useful as a detection system(see, for example, International PCT application No. WO98/20019; see,also, Whittal et al., Anal. Chem. 70:5344-5347 (1998), describing theuse of MALDI-TOF mass spectrometry for the analysis of proteins isolatedfrom single cells).

A charge coupled device (CCD) camera can be useful for detecting andimaging fluorescent, chemiluminescent or radionuclide labeled materials.Such a detection system has been adapted to analysis of reactions onmicrochips, and can allow the detection of thousands of bindingreactions performed on a microchip (see Eggers and Ehrlich, Hematol.Pathol. 9:1-15 (1995); see, also, Eggers et al., Biotechniques17:516-525 (1994). Using a CCD detecting system, biopolymers, which canbind an appropriately labeled material, or reagents that can bind anappropriately labeled material such as a fluorescently labeledbiopolymer, are immobilized directly on the pixels of a CCD, and,following a reaction as desired, bound labeled materials are detected atthe specific pixel location. The signal obtained from a CCD can bedisplayed, if desired, and can allow a quantitative determination, forexample, of binding events. Furthermore, since the signal obtained usinga CCD detector is received in real time, the signal can be used as anindication of the extent of a reaction, and can be interfaced with theliquid dispensing system to cause an amount of a liquid to be dispensed,for example, to terminate the reaction, or to change the conditions ofthe reaction such that a second reaction can be performed at theparticular target site.

Where a substrate, or a product of a reaction to be detected, is labeledusing, for example, a luminescent, fluorescent or chemiluminescentlabel, a fiber optic system can be used as a detection system (see, forexample, Clark et al. CHEMTECH 28:20-25 (1998)). Fiber optics areparticularly useful because their small size permits the monitoring ofindividual target sites, for example, in an array of target sites on amicrochip. In addition, a fiber optic system can provide the additionalfunction of monitoring the level of a liquid, as disclosed above. Otherdetection systems including laser scanners (Cheung et al., Nature Genet.21:1519 (1999), capillary electrophoresis (Hadd et al., Anal. Chem.69:3407-3412 (1997), and epifluorescence microscopy (Fodor etal.,Science 251:767773 (1991) have been adapted to microchip devices andglass slides and can be used in an open system as disclosed herein.

Methods

Methods for maintaining a volume of a liquid in an unsealed environmentalso are provided. Such a method can be performed in an open system, forexample, by determining the temperature of a solid support having atarget site, which can contain the volume of liquid; and, based on thetemperature, dispensing at the target site an amount of liquid requiredto maintain the volume of the liquid. The amount of liquid to bedispensed can be determined, for example, using a computer algorithmthat can calculate, based on various parameters, including thetemperature of the support, the chemical nature of the liquid, and thevolume to be maintained, the amount of liquid that evaporates from thesite, such that the amount of liquid dispensed corresponds to the amountof liquid that evaporates. Alternatively, the volume can be monitored,for example, by a microscopic examination, using an appropriate opticalsystem, or by a video imaging technique, such that, as the volume of aliquid at a target site decreases, liquid can be dispensed to maintainthe volume within acceptable parameters. In another embodiment, thevolume can be monitored by tracking the meniscus of the liquid anddetermining when the meniscus decreases below a defined point. Suchmonitoring can be performed by detecting a change in a circuit due to adecrease in the amount of liquid below a level required to maintain thecircuit, or by a change in the quality of light being transmitted intothe liquid.

Methods for performing a reaction in an unsealed environment also areprovided. Such a method can be performed, for example, by determiningthe temperature of a solid support, which includes a target sitecontaining a volume of the reaction mixture; and dispensing into thereaction mixture an amount of liquid required to maintain the volume.Such a method is particularly useful where the reaction mixture has avolume of a few microliters or less, generally a volume of about 20microliters or less, and particularly about 500 nanoliters or less. Thedisclosed methods also are useful for performing submicroliter reactionsat temperatures where the vapor pressure of a liquid in the reactionmixture is undesirably high, for example, about 2.5 kiloPascals (kPa) orgreater, particularly about 5 kPa or greater, or about 10 kPa orgreater, such that evaporation of the liquid can substantially changethe volume of the reaction mixture and adversely affect the reaction.

The disclosed methods are useful, for example, for performing a reactionin an aqueous environment at a temperature greater than about 22° C.(room temperature; RT; 72° F.), particularly at a temperature about 37°C. or greater, where the vapor pressure for water is 2.6447 kPa at 22°C., 6.2795 kPa at 37° C., and 84.529 kPa at 95° C. (“Handbook ofChemistry and Physics” 75th ed. (CRC Press, Inc., 1994); see pages 6-15to 6-17; see, also, 6-77 to 6-108; and 15-43 to 15-49). As such, thedisclosed methods can be used, for example, to perform various chemical,physical and biological reactions such as synthesis of a combinatoriallibrary, a mammalian cell transfection, or a polymerase chain reaction.

A method for maintaining a volume, particularly a submicroliter volume,of a reaction mixture on a solid support in an unsealed environment canbe performed by determining the rate of evaporation of a liquid from thereaction mixture; and dispensing into the reaction mixture an amount ofliquid that corresponds to the amount of liquid that evaporates, therebymaintaining the volume of the reaction mixture.

Such a method can be useful, for example, where the reaction mixturecontains a biopolymer, which can be a substrate or a product of thereaction. Following a reaction using a method as disclosed herein, thebiopolymer or products of the biopolymer can be detected, eitherdirectly or indirectly. As such, the disclosed methods are useful fordetermining the sequence of a biopolymer, for synthesizing a biopolymerfrom monomeric subunits, and for detecting the presence of a particularbiopolymer, for example, in a biological sample. Various methods fordetecting a biopolymer are exemplified herein and other methods, whichare selected based, in part, on the particular type of biopolymer, arewell known to those in the art.

A method as disclosed herein can be useful for essentially any type ofreaction, including, for example, where the substrate is a biopolymer, abiological reaction such as an enzyme-mediated polymerization, ligation,cloning, or a degradation reaction; a physical reaction such as anucleic acid hybridization, the binding of a nucleic acid regulatoryelement by a particular nucleic acid binding polypeptide, orhomodimerization or heterodimerization of polypeptides; or a chemicalreaction such as a chemical labeling reaction, chemical synthesis of thebiopolymer; or a chemical cleavage of the biopolymer, for example,cyanogen bromide cleavage of a polypeptide at a methionine residue ordimethylsulfate cleavage of a carbohydrate end group. The reaction alsocan be a chemical synthesis reaction, for example, synthesis of acombinatorial library of small molecules or of biopolymers; or can be ahydrolysis reaction such as a polysaccharide hydrolysis reaction.

A method as disclosed herein also is useful for a reaction involving aliving cell. For example, the reaction can be a method of introducing arecombinant nucleic acid molecule, which can be contained in a vector,into a host cell in order to produce copies of the recombinant nucleicacid molecule or to express a polypeptide encoded thereby. Such apolypeptide can be isolated, if desired, or can be expressed for thepurpose of providing an advantage to the cell expressing thepolypeptide. The reaction also can involve contacting a cell with aphysical, chemical or biological agent in order to identify a change ingene expression in the cell, for example, by imposing a heat shock onthe cells, or by contacting the cells with a putative medicament. Anopen system as disclosed herein is useful for such reactions because itallows precise control of the reaction conditions, particularly theability to maintain the reaction at a predetermined volume. Furthermore,because the reactions can be performed in very small volumes, studiesinvolving only one or a few cells can be performed (see, for example,Clark et al. CHEMTECH 28:20-25 (1998)).

Sequencing Reactions

The methods disclosed herein can be used for a biopolymer sequencingreaction. For example, the biopolymer can be a polynucleotide, which canbe sequenced using the Maxam-Gilbert chemical cleavage method, or anenzymatic reaction such as the Sanger-Coulson chain termination methodor an exonuclease cleavage method. A biopolymer sequencing reaction suchas the Maxam-Gilbert method or Sanger-Coulson method conveniently can beperformed, for example, on a microchip, in which a number of reactions,including the four (or five) base specific reaction, can be performed inparallel on one or more polynucleotides, or a single base reaction canbe performed on a number of different polynucleotide sequences. Suchmethods of polynucleotide sequencing result in the production of nestedfragments of the polynucleotide, which can be detected using variousmethods, particularly mass spectrometry, including matrix-assisted laserdesorption/ionization time-of-flight (MALDI-TOF) mass spectrometry orcapillary electrophoresis.

In a chain termination sequencing reaction, for example, each reactionmixture is an aqueous solution, containing a polymerase, four nucleosidetriphosphates, and a chain terminating nucleoside triphosphate; thewater in the reaction mixture is susceptible to evaporation during thereaction, which generally is performed at a temperature of about 37° C.Particularly where the reaction is performed in a submicroliter volumeon a solid support in an unsealed environment, the water can evaporatealmost completely in a short period of time and, even where the reactionmixture does not evaporate to dryness, the loss of water volume cansubstantially alter the kinetics of the polymerase reaction, includingthe fidelity of nucleotide incorporation, and produce spurious results.A method as disclosed herein avoids such a problem by dispensing intothe reaction mixture an amount of water that corresponds to the amountof water that evaporates during the reaction.

In the Maxam-Gilbert sequencing method, four or five separate reactionscan be performed, each of which is performed under different conditions,with different liquids, including water, dimethyl sulfate, piperidine,and hydrazine (see Sambrook et al., “Molecular Cloning: A laboratorymanual” 2nd ed. (Cold Spring Harbor Laboratory Press 1989), pages13.11-13.13). For example, a sugar-phosphate cleavage reaction of themodified bases is performed using 1 M piperidine in water at 90° C.(vapor pressure of piperidine=84.1 kPa at 100° C.; and of water=101 kPaat 100° C.). As such, a method as disclosed herein, can be used tomonitor the evaporation of piperidine and of water during thesugar-phosphate cleavage reaction and can allow an amount of each liquidto be dispensed to the target site during the reaction. The relativeamount of each such liquid to be dispensed can be calculated, forexample, based on the concentration of each liquid in the reaction andthe vapor pressures of the liquids.

A polynucleotide also can be sequenced by an exonuclease reaction usinga method as disclosed herein. For example, a multiplex exonucleasesequencing reaction can be performed on polynucleotides as disclosed inU.S. Pat. No. 5,622,824 and U.S. Pat. No. 5,851,765, wherein massdifferentiated nucleic acid molecules containing mass modifiednucleotides are prepared, immobilized to a target site, and contactedwith an exonuclease. A number of exonuclease sequencing reactions can berun in parallel, for example, on a microchip, and the concentration ofexonuclease, pH, and time of incubation can be varied in the differentreaction mixtures to produce a desired range of degradation products.Time of incubation can be adjusted, for example, by maintaining thetemperature of each target site at 4° C., then at predetermined times,adjusting the temperature of one or more target site to 37° C. Thevolume of each reaction mixture is maintained at a predetermined volumethroughout the procedure. At the appropriate time, all of the reactionsare terminated, for example, by removing the liquid and dissolvedreagents from the target sites. The immobilized, exonuclease degradedmass differentiated nucleic acid molecules remain immobilized to thechip and are available for detection, for example, by mass spectrometry.

A polypeptide also can be sequenced using a chemical degradation method,for example, the Edman degradation method, which utilizesphenylisothiocyanate to sequentially cleave amino acids from the aminoterminus of a polypeptide, or an enzymatic degradation reaction using anexopeptidase such as a carboxypeptidase, which sequentially cleavesamino acids from the carboxy terminus of a polypeptide, or anaminopeptidase, which sequentially cleaves amino acids from the aminoterminus of a polypeptide.

The sequentially released amino acids or nested fragments of thepolypeptide can be detected. Nested fragments of a polypeptide can beproduced conveniently by performing a number of reactions in parallel ona microchip, where the polypeptides are reversibly immobilized to thesolid support using, for example, photocleavable linkers, chemicallycleavable linkers, or the like. After the reactions are complete and theimmobilized fragments have been washed, they can be detected in situ, orcan be released from the support for detection.

The methods as disclosed herein also can be useful for reactions inwhich a biopolymer is cleaved into smaller, but not necessarilymonomeric, fragments. For example, a polynucleotide can be cleaved,partially or completely, using a restriction endonuclease or basespecific endonuclease such as various RNA endonucleases. Similarly, apolypeptide can be cleaved into fragments using, for example, cyanogenbromide, which cleaves at methionine residues, or any of variousendopeptidases such as trypsin and chymotrypsin. The fragments producedthen can be detected to facilitate determining the order of thefragments within the larger polynucleotide or polypeptide. The methodsdisclosed herein also are applicable where the biopolymer is acarbohydrate, glycoprotein, proteoglycan or lipid, which is to besequenced.

Synthesis Reactions

The disclosed methods for maintaining a volume, particularly asubmicroliter volume, of a reaction mixture also are useful for abiopolymer synthesis reaction. For example, the biopolymer can be apolynucleotide, and the synthesis reaction can be a chemical synthesisreaction or an enzymatic synthesis reaction using a polymerase (see, forexample, S. M. Hecht, ed. “Bioorganic Chemistry: Nucleic acids” (OxfordUniv. Press 1996)). Chemical synthesis of a polynucleotide can beperformed using any of various methods, including the phosphotriester,phosphoamidate and H-phosphonate method (Hecht, ed. “BioorganicChemistry: Nucleic acids” Oxford Univ. Press 1 996, pages 36-74), andutilizes various organic solvents, including, for example, acetonitrile,which has a vapor pressure of about 11.8 kPa at 25° C. and, therefore,is more susceptible to evaporation than water. As such, the disclosedmethods are particularly useful for chemical polynucleotide synthesisreactions, since the volume of the reaction mixtures can be maintainedat a predetermined volume throughout the reaction period.

An enzymatic synthesis reaction, in comparison, is performed in anaqueous solution. The biopolymer can be a ribonucleic acid, and thepolymerase can be an RNA dependent RNA polymerase or an RNA dependentDNA polymerase. Where the polymerase is an RNA dependent DNA polymerase,the enzymatic synthesis reaction also can include a DNA dependent DNApolymerase, for example, a reverse transcription-polymerase chainreaction (RT-PCR).

A polynucleotide can be synthesized, for example, by PCR. In addition tothe substrate polynucleotide and a polymerase, which can be a DNApolymerase or RNA polymerase, other components of a PCR reaction includenucleoside triphosphates, which can be deoxyribonucleotides,ribonucleotides or analogs thereof, and a set of primers, including aforward primer and a reverse primer. Nested PCR reactions also can beperformed, in which case a second set of primers, which specificallyhybridize to the first amplification product, are a component of thereaction. A primer can be any oligonucleotide, including anoligonucleotide containing oligonucleotide mimetics, such as PNA(protein nucleic acid formed by conjugating bases to an amino acidbackbone, which render the base sequence less susceptible to enzymaticdegradation; see, e.g., Nielsen et al. (1991) Science 254:1497),portion(s), provided that the nucleotide at the 3′ end of such a primeris linked to the oligonucleotide by a phosphodiester bond, or the like,such that extension of the primer from the 3′ end can occur. Thedisclosed methods of maintaining a reaction mixture in an unsealedenvironment at a predetermined volume are particularly valuable forperforming a PCR reaction, since PCR utilizes a number of differentsteps, performed at different temperatures, including temperatures ashigh as 95° C. Thus, the disclosed methods provide a means to monitorthe reaction volume at different times during the PCR reaction anddispense liquid, generally water, to the target site in order tomaintain the reaction at a predetermined volume.

The disclosed methods for synthesizing a biopolymer in a submicroliterreaction also can be used to synthesize a polypeptide. The disclosedmethods also can be used to synthesize a carbohydrate, a glycoprotein, aproteoglycan or a lipid in a submicroliter reaction mixture.

Diagnostics

Genetic factors may contribute to virtually every human disease,conferring susceptibility or resistance, affecting the severity orprogression of disease, and interacting with environmental influences.much of current biomedical research, in the public and private sectors,is based upon the expectation that understanding the geneticcontribution to disease will revolutionize diagnosis, treatment, andprevention. Analysis of DNA sequence variation is becoming anincreasingly important source of information for identifying the genesinvolved in disease and in normal biological processes such asdevelopment, aging and reproduction.

Genomic research has identified several types of DNA sequencevariations, including insertions and deletions, differences in the copynumber of repeated sequences, and single base pair differences such assingle base pair deletions, termed single nucleotide polymorphisms(SNPs). SNPs occur with a frequency of about 1% (or about 1 millionSNPs) in the human genome and serve as markers of regions in the humangenome. While biological processes and diseases are caused or influencedby complex interactions among multiple genes and environmental factors,many alleles associated with health problems may have low penetrance,meaning that only a few of the individuals carrying them will developdisease. SNPs better identify regions important for mapping anddiscovering the genes associated with common diseases. In addition totheir frequency, SNPs are attractive candidates as genetic markers dueto their stability, generally having much lower mutation rates, and theamenability of automating the analysis of such sequences, therebyallowing large scale genetic analysis.

The screening and scoring of the million or so SNPs and the genetic locipredicted make up the human genome largely is dependent on thescientific community's ability to reduce the cost of such analysis. Overthe past few years, scientists have begun to develop methods such asmultiplexing, which allow the analysis of more than one genetic locusper sample. Further development of such methods has led to the use ofnanotechnology, which has miniaturized sample preparation andbiochemical reactions, allowing significant cost savings and movement ofDNA analysis toward automation. The open systems and methods disclosedherein provide a substantial step forward in adapting nanotechnology tothe analysis of biopolymers, including in situ biopolymer synthesis andsequencing, and diagnostic assays such as oligonucleotide based primerextension reactions and PCR.

Methods of Detecting the Presence of a Biopolymer

The disclosed methods of performing a reaction in a submicroliter volumein an unsealed environment are useful for detecting the presence of thebiopolymer, which can be in a biological sample, because of the abilityto perform such assays with only a small amount of sample. As such, thedisclosed methods are particularly useful for performing clinicaldiagnostic assays.

The biopolymer can be, for example, a polynucleotide, which isimmobilized to the solid support and detected by identifying a detectoroligonucleotide that hybridizes to the biopolymer. The detectoroligonucleotide can be a peptide nucleic acid. The method can beperformed with a plurality of reaction mixtures, wherein one or more ofthe plurality of reaction mixtures contains a biopolymer, which can beimmobilized to the solid support. In such a method, the solid supportcan be a microchip, and the plurality of reaction mixtures is present inan array on the microchip.

Any component of the reaction mixture can be detected, as desired,including, for example, the biopolymer, which detected directly orindirectly. For example, where the biopolymer is a polynucleotide, itcan be detected by identifying an amplification product produced fromthe polynucleotide, or by identifying a reagent such as anoligonucleotide that binds specifically to the biopolymer. Theoligonucleotide can include a PNA portion, and can be an oligonucleotideprimer that has been extended due to the activity of a polymerase. Wherethe biopolymer is a polypeptide, the reagent can be a second polypeptidethat binds specifically to the first polypeptide, for example, anantibody.

The disclosed methods of performing a reaction in a submicroliter volumein an unsealed environment also can be used to examine a polynucleotideusing a primer extension reaction. The primer extension reaction can becompetitive oligonucleotide single base extension; primer oligo baseextension (PROBE); loop-PROBE; or telomeric repeat amplificationprotocol, and the primer can be any oligonucleotide, including, forexample, an oligonucleotide containing a peptide nucleic acid portionand having a 3′ terminus that is a substrate for a polymerizationreaction (see, e.g., International PCT application No. WO98/20019).

Such methods provide diagnostic assays, including assays for detectingthe presence of, or predisposition to a disease or condition. Such adisease or condition can be a genetic disease, for example, Huntington'sdisease, prostate cancer, Fragile X syndrome type A, myotonic dystrophytype l, Kennedy's disease, Machado-Joseph disease, dentatorubral andpallidolyusian atrophy, and spino bulbar muscular atrophy; or thecondition can be aging, which can be identified by examining the numberof nucleotide repeats in telomere nucleic acid from a subject. Thedisease or condition also can be associated with a gene such as genesencoding BRCA1, BRCA2, APC; a gene encoding dystrophin, β-globin, FactorIX, Factor VIIc, ornithine-d-amino-transferase, hypoxanthine guaninephosphoribosyl transferase, or the cystic fibrosis transmembranereceptor (CFTR); or a proto-oncogene.

The methods as disclosed herein also are useful for detecting singlenucleotide polymorphisms (SNPs), which occur with a frequency of about1% (or about 1 million SNPs) in the human genome and serve as markers ofregions in the human genome. SNPs can identify regions important formapping and discovering the genes associated with common diseases, andare attractive candidates for analysis because of their frequency in thehuman genome and because of their relatively low mutation rates. Assuch, analysis of SNPs is amenable to automation.

A method as disclosed herein can be used to genotype a subject bydetermining the identity of one or more allelic variants of one or morepolymorphic regions in one or more genes in the subject. For example,the one or more genes can be associated with graft rejection and theprocess can be used to determine compatibility between a donor and arecipient of a graft. Such genes can be MHC genes, for example.Genotyping a subject using a process as provided herein can be used forforensic or identity testing purposes and the polymorphic regions can bepresent in mitochondrial genes or can be short tandem repeats.

A disclosed method also can be used to determine whether a subject isinfected with an infectious organism such as a virus, bacterium, fungusor protist. A process for determining the isotype of an infectiousorganism also is provided. Thus, depending on the sequence to bedetected, the methods disclosed herein are useful, for example, todiagnose a genetic disease or chromosomal abnormality; a predispositionto or an early indication of a gene influenced disease or condition, forexample, obesity, atherosclerosis, diabetes or cancer; or an infectionby a pathogenic organism, for example, a virus, bacterium, parasite orfungus; or to provide information relating to identity, heredity orcompatibility using, for example, mini-satellite or micro-satellitesequences or HLA phenotyping.

Libraries

The disclosed methods for performing a reaction in an unsealedenvironment are useful for producing libraries containing diversepopulations of molecules, including chemical or biological moleculessuch as simple or complex organic molecules, peptides, proteins,peptidomimetics, glycoproteins, lipoproteins, polynucleotides, and thelike. Libraries containing such molecules and methods of makinglibraries, such as combinatorial libraries, are known in the art (see,for example, Huse, U.S. Pat. No. 5,264,563; Gallop etal., J. Med. Chem.37:1233-1251 (1994); Gordon et al., J. Med. Chem. 37:1385-1401 (1994);Blondelle et al., Trends Anal. Chem. 14:83-92 (1995); Eichler andHoughten, Molec. Med. Today 1:174-180 (1995); York et al., Science274:1520-1522 (1996); Gold et al., Proc. Natl. Acad. Sci., USA 94:59-64(1997); Gold, U.S. Pat. No. 5,270,163, issued Dec. 14, 1993).

Libraries, such as combinatorial libraries, can contain as many as 10¹⁴to 10¹⁵ different molecules, and typically contain on the order of10³-10⁶. The diverse molecules in a combinatorial library can be based,for example, on a known molecule, such as known pharmacophore scaffold,which is being diversified to find a new, but similar molecule havingmore desirable characteristics such as better solubility or the abilityto be administered orally or bioactivity. Of course, the diversemolecules also can be randomly designed molecules, which can be screenedfor a desirable characteristics.

The methods herein allow libraries of diverse molecules to be producedin an open system and, if desired, using a single tube format. Inaddition, the diverse molecules produced can be screened in situ, forexample, where the library is a library of diverse antibodies, they canbe screened using the appropriate antigen and a suitable binding assay;or where the library is a library of diverse drugs, which are based on adrug that can inhibit a protein-protein interaction involved, forexample, in a metabolic pathway associated with a disease, the diversedrugs can be screened by contacting a drug with the proteins andmonitoring the association of the proteins. A screening assay provides asimple means for identifying those agents in the library that have adesirable property. Thus, a screening assay can be performed followingpreparation of a combinatorial library, and the entire process can beautomated using an open system as disclosed, thereby allowing for highthrough-put screening assays.

The following examples are included for illustrative purposes only andare not intended to limit the scope of the invention.

EXAMPLE 1 Unsealed Nanoliter PCR Monitored by Fluorescence EmergyTransfer Assay in a Single Well Procedure

This example demonstrates that a PCR amplification performed in an opensystem can be detected online by increasing fluorescence using afluorescence energy transfer assay, the TaqMan™ assay (Nucleic AcidsRes. 25:1999-2004 (1997)).

The TaqMan™ fluorescent assay uses an oligonucleotide probecomplementary to an internal segment of the target DNA to be amplified.The probe is labeled with two fluorescent moieties. As a result of theoverlap between the emission and excitation spectra of the twofluorescent moieties, one moiety quenches the emission of the othermoiety. The presence of this probe during PCR allows the amplificationprocess to be monitored. The probe hybridizes to the target DNA duringthe PCR process and becomes susceptible to degradation by the 5′nuclease activity of Taq polymerase, which is specific for DNAhybridized to the template. As a result of the nucleolytic degradation,the two fluorescent labels are no longer in proximity, thereby reducingthe quenching and increasing the intensity of the emitted light. As aresult, measurement of fluorescence during amplification permitsreal-time monitoring of the PCR yield.

A TaqMan™ kit (Applied Biosystems, Foster City Calif.) contains thefollowing components: human genomic DNA at a concentration of 10 ng/μl,forward and reverse primers specific for the human β-actin gene: forwardprimer 5′-TCACCCACACTGTGCCCATCTACGA-3′ (SEQ ID No. 1); and reverseprimer (5′-CAGCGGAACCGCTCATTGCCAATGG-3′ (SEQ ID No. 2); a dualfluorophore-labeled probe:5′-(6-carboxyfluorescein)-ATGCCC-(6-carboxytetramethylrhodamine)-CCCCCATGCCATCCTGCGT-3′(SEQ ID No. 3) complementary to the β-actin specific PCR product. Thereaction mixture contains 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 0.01%gelatin, 1 mg/ml bovine serum albumin (BSA), 3.5 mM MgCl₂, 200 μM ofeach dNTP, 300 μM of the forward and reverse amplification primers, 200μM of the dual fluorophore-labeled probe, 0.5 Units Taq polymerase, 0.1Units anti-Taq antibody and 5 ng of template DNA in μl total volume. PCRis performed using the following cycling conditions: 40 cycles of 94° C.for 10 seconds, 54° C. for 5 seconds, and 72° C. for 15 seconds.

PCR was performed on a modified 2 channel NANO-PLOTTER pipetting devicetype NP1c (GeSim, Dresden Germany). This fluid dispenser device has anxyz table to move and dispense liquids from a piezo electric pipette.The pipettes are connected to pump system (diluters) to fill thepipettes with liquid in the nanoliter scale from a microliter platedeposited on the z-table of the pipetting device. The other end of thediluters are connected to a reservoir which contains system liquid, forexample, 50 ml ultrapure water. Valve settings in the diluters allows tobypass system liquids to the piezo electric pipettes.

The NANO-PLOTTER (GeSiM, Germany), discussed above, is modified so thatthe target sites includes a 22W Peltier heating/cooling element, orother such element, which is controlled by a programmable thermoelectrictemperature controller LFI-3526 (Wavelength Electronic, Inc., BozemanMont.). Modification with reference to the NANO-PLOTTER refers tocoupling the device with a heating element to heat the liquid before itis dispensed onto the chip support. This can be effected by heating ofthe microtiter dish or heating the source of the water, such as thereservoir. Temperature is measured using a PT 500 type F3132 (NewportElectronics, Deckenpfronn Germany). System liquid is heated to targettemperature directly in the pipette to keep the temperature gradientbetween reaction liquid on the target and subsequently added systemliquid zero.

The reaction is deposited on two different positions on a microchipsupport (which will be sold under the trademark Spectrochip™ bySequenom, Inc., San Diego Calif.), which has a hydrophobic surface withhydrophilic target sites for retaining aqueous reaction mixtures. Thechip support contemplated in this example, includes two modifiedhydrophobic positions that allow the reaction liquid to grow only in thez-direction. One position on the chip is used to monitor the reactionusing a fiber optic set very close to the reaction position. The fiberoptic is connected to a photomultiplier to convert the fluorescencesignal to an electric signal. The bandpass filter is placed between thefiber optic and the photomultiplier to cut-off the exciting wavelength.Fluorescence is excited with a 15 mW argon laser and detected throughthe recommended bandpass filter(Applied Biosystems).

The second position on the chip (dummy position) is used to monitor theliquid loss due to evaporation. An inter digital array is set very closeto the dummy position in order to monitor drop size by capacitymeasurements. The reaction mix and the dummy drop are kept constantduring each PCR cycle by adjusting the dispensing frequency of thesystem liquid. Since the capacity can be changed in both directions,when the drop volume increased or decreased, the frequency of addedreservoir liquid is related to the cycle program, so that the slope offrequency is negative when the cycle temperature is decreased and ispositive when the cycle temperature is increased. The whole system canbe covered and darkened to reduce background effect while obtaining thefluorescence signal.

Five nl (25 drops) of reaction mix and dummy liquid are transferred froma microtiter plate onto a cooled (5° C.) 2-position silicon SpectrochipTmicrochip. This cooled trap is used to determine the initial drop size,without evaporation. Once the initial drop size is determined the PCRprogram is started. During PCR, the pipettes replace evaporated waterfrom the system liquid based on the measured capacity and fluorescencesignal, as obtained online. Liquid loss also can be monitored by laserscan microscopy, where the whole dummy drop is irradiated by a He-Nelaser (632 nm) and monitored (Fraunhofer Inst., Erlangen Germany). Eachdeviation of drop size is recognized and liquid is dispensedaccordingly.

For a 97 tube procedure, including 96 reactions and one dummy reaction,97 very small laser diodes (2×2 mm) are placed under the target sites toexcite fluorescence in the reaction volume from behind. An advantage ofthis system is that each of the 97 reactions can be monitored at thesame time without scanning the argon laser over the whole chip. Thereaction volume is dispensed onto a glass pad, and the laser diode ismounted under the Peltier element to provide the diode with its workingtemperature, because the storage temperature of a laser diode is lowerthan the temperature used in a reaction such as PCR and, therefore,cannot be placed between the target and the peltier element. Thus, thePeltier element is constructed with holes to allow passage of thefocused laser beam. A 97 diode array also can be placed at a differentlocation.

The same experiment, in a 97 tube or 385 tube format, can be performedusing two different blocks, containing active and passive piezo electricpipettes. The active block contains 1, 4 or 8 pipettes and the includesa system to transfer samples from a microliter plate. The passive blackcontains 97 (385) pipettes (including the dummy tip) with the samemeasurements as the SpectrochipT microchip. Each piezo electric pipetteof the active/passive block is addressable with its own piezo electricdispensing parameters. With the passive block, system liquid is addedsimultaneously onto each of the 97 (385) positions of the chip.

EXAMPLE 2 Unsealed Nanoliter PCR Prior to Maldi-ms Analysis in a SingleTube Procedure

This example demonstrates that PCR products produced in an unsealedenvironment can be detected using matrix assisted laser desorptionionization (MALDI) mass spectrometry.

PCR is performed using pACT template, which is a pUC derivativeharboring a 389 bp insert of human β-actin cDNA, biotinylated primerBAct2 d(bio-GAC TGA CTA CCT CAT GAA GAT CC) (SEQ ID No. 4) andnon-biotinylated primer Act4 d(GAA GCT GTA GCC GCG CTC GG) (SEQ ID No.5). The reaction mix contains 5 μl 10×PCR buffer (200 mM Tris-HCl (pH8.75), 100 mM KCl, 100 mM (NH₄)₂SO₄, 20 mM MgSO₄, 1% Triton X-100, 1 mgBSA), a final concentration of 200 μM of each dNTP, 10 pmol biotinylatedprimer, 100 pmol non-biotinylated primer, 2.5 Units Pfu DNA polymerase(Stratagene; La Jolla Calif.), 0.25 ng of template, and water to a finalvolume of 50 μl. Amplification cycles were as follows: 30 cycles of 94°C. for 30 seconds, 65° C. for 30 seconds, and 72° C. for 30 seconds.

PCR was performed using a 1 channel NANO-PLOTTER pipetting device typeNP1C (GeSim, Dresden Germany) modified with a heating element asdescribed herein. This pipetting device has an xyz table to move anddispense liquids from a piezo electric pipette. The pipette is connectedto a pump system (diluter) to fill the pipette with liquid in thenanoliter scale from a microliter plate deposited on the z-table of thepipetting device. The other end of the diluter is connected to areservoir that contains system liquid, for example, 50 ml ultrapurewater. Valve settings in the diluter allow the bypass of system liquidto the piezo electric pipette. It is modified for use herein such thatthe target site (site to which liquid is dispensed) of the pipettingdevice has a 22W peltier heating/cooling element that is controlled by aprogrammable thermoelectric temperature controller LFI-3526 (WavelengthElectronic, Inc., Bozeman Mont.). Temperature is measured using a PT 500type F3132 (Newport Electronics, Inc., Deckenpfronn Germany).

System liquid is heated to target temperature direct in the pipette tokeep the temperature gradient between reaction liquid on the target andsubsequently added system liquid zero. The reaction mix is deposited ontwo different positions on a Spectrochip™ microchip, which contains twohydrophilic modified positions. These target sites are surrounded by ahollow of black silicon containing a dense “forest” of 10 μm highneedles. Due to the sharp edge between the target site and the hollow,the reaction volume grows only in the z-direction to a defined volume.Under the position where PCR occurs, a small magnet is mounted tocapture paramagnetic beads, for example, streptavidin coatedparamagnetic beads.

The second position (dummy position) is used to monitor the liquid lostdue to evaporation. An inter digital array is set very close to thisposition to monitor drop size based on capacity measurement. Thereaction mix and the dummy drop are kept constant during each PCR cycleby adjusting the dispensing frequency of the system liquid (see Example1).

Five nl (25 drops) reaction mix are transferred from a microtiter plateonto the cooled (5° C.) Spectrochip™ microchip. The cooled trap is usedto determine the initial drop height without evaporation. Once themixture is deposited, the initial height of the reaction liquid ismeasured and PCR program was started. Following PCR, the Spectrochip™microchip is cooled to 5° C. to trap the reaction liquid. In a firststep after PCR, the piezo electric pipette transfers 10 nl streptavidindynabeads (M-280) from a microtiter plate into the reaction mixture tocapture PCR product. In a second step, the piezo electric pipette isused to flood the target area with 1 μl of 0.07 M ammonium citratesolution to rinse the reaction mix into the hollow. The washing step isrepeated once. In a third step, the PCR product is denatured from thebeads using ammonia at room temperature (RT). Thus, the target wasadjusted to RT and the piezo electric pipette picks up 10% ammonia andtransferred 50 nl onto the target site. In the fourth step, the targetis cooled to 5° C. and the denatured PCR product is redissolved with 3nl ultrapure water. In the last step, 6 nl matrix is added into theliquid PCR product, while again adjusting the target temperature to RTto obtain optimal crystallization. The SpectroChip™ support then istransferred into a mass spectrometer (Bruker/Sequenom, Germany), whichallows automated measurement of the nanoliter reactions.

The reaction also can be monitored using a He-Ne laser, and can beperformed using active and passive piezo electric pipettes as disclosedin Example 1. Using a 4 channel NANO-PLOTTER pipetting device, the last4 steps as described in Example 1 can be reduced to 2 steps, including afirst step, wherein pipette 1 contains the beads and pipette 2 containsthe rinse liquid, and a second step, wherein pipette 3 contains theammonia and pipette 4 contains the matrix. Utilizing two steps preventsthe ammonia from evaporation and the matrix from crystallization.

EXAMPLE 3 Unsealed Nanoliter Cycle Sequencing Prior to Maldi-ms Analysisin a One Well Procedure

This example demonstrates that cycle sequencing can be performed in asingle well and the reaction product subsequently can be analyzed byMALDI-MS. Using the open method, no cover or sealing is used to preventevaporation during the cycling program for the DNA sequencing reaction.

Reactions were performed essentially as described previously (see vanden Boom et al., Anal. Biochem.; van den Boom et al., J.B.B.M. standardcycle sequencing paper; Koster et al., Nature Biotech.; Little et al.,Anal. Chem. 69:4540-4546 (1997)). DNA sequencing of PCR products areamplified off-line on a microchip without sealing. Target DNA to besequenced was amplified from genomic DNA or cDNA using a biotinylatedprimer. The corresponding PCR product was stored in a microtiter plateaccessible for the piezoelectric pipette. PCR product can be placed oneach position of a 96 Spectrochip™ support, thus allowing a series ofcycle sequencing reactions to be performed according to a primer walkingstrategy, which yields the full sequence of the PCR product.

Sequence analysis is performed by a thermal cycled reaction using ashort oligonucleotide primer complementary to the biotinylated PCRstrand, a DNA polymerase and a sequencing nucleotide mix in a bufferedreaction system. After completion of the reaction, the biotinylated PCRtemplate strand is immobilized to streptavidin coated magnetic beads.The sequencing ladder that hybridizes to this strand is co-immobilizedand can be separated form reaction components. After a washing step, thesequencing ladder is recovered from the streptavidin beads bydenaturation. In a subsequent step, the sequence ladder can be massanalyzed directly from the SpectroChip™ support using a Bruker/SequenomMALDI-TOF MS.

The following reagents were used: p53 specific PCR product:amplification primers d(CTGCTTGCCACAGGTCTC) (SEQ ID No. 6) andd(biotin-CACAGCAGGCCAGTGTGC) (SEQ ID No. 7) were targeted against exon 7of the p53 gene. PCR was performed according to standard proceduresusing 10 pmol of forward primer and 6 pmol of biotinylated reverseprimer. Sequencing primer specific for the exon 7 PCR product wasd(gaggcccatcctcacc) (SEQ ID No. 8). The cycle sequencing reactionmixture contained 10 mM Tris-HCl (pH 8.3), 0.01% gelatin, 1 mg/ml BSA,3.5 mM MgCl₂, 200 μM of each dNTP, 300 μM of sequencing primer, 1 UnitThermoSequenase™ and 5 ng of p53 PCR product in a 10 μl total volume.M-280 streptavidin coated paramagnetic beads also were used. Cyclesequencing was performed using the following cycling conditions: 30cycles of 94° C. for 10 seconds, 54° C. for 5 seconds, and 72° C. for 15seconds.

Liquid handling was performed using a 2 channel NANO-PLOTTER pipettingdevice type NP1c (GeSim) modified with heating element. As noted above,this device contains an xyz table to move and dispense liquids from apiezoelectric pipette. The pipettes are connected to pump systems(diluters) to fill the pipettes with liquid in nanoliter volumes from amicroliter plate deposited on the z-table of the pipetting device. Theother end of the diluters is connected to a reservoir containing thesystem liquid, 50 ml ultrapure water. Valve settings in the dilutersallow to bypass system liquids to the piezo electric pipettes. Thetarget site of the modified NANO-PLOTTER pipetting device has a 22WPeltier heating/cooling element controlled by a programmablethermoelectric temperature controller LFI-3526. Temperature is measuredvia PT 500 type F3132 (see Examples 1 and 2).

System liquid is heated to target temperature directly in the pipette tokeep the temperature gradient between reaction liquid on the target andsubsequently added system liquid zero. The reaction liquid is depositedon two different positions on a microchip support (e.g., a SpectroChip™chip), which contains 2 hydrophilic modified positions (see Example 2).Under the position where the reaction takes place, a small magnet ismounted to capture paramagnetic beads. A second dummy position isincluded as described in Examples 1 and 2.

Five nl reaction mixtures are transferred from a microtiter plate onto acooled (5° C.) Sequenom 97 position silicon SpectroChip™ microchip andthe initial height of the reaction liquid is determined (see Examples 1and 2). After the cycled sequencing reaction is completed, theSpectroChip™ microchip is cooled to 50° C. to trap the reaction liquid.

In a first step the piezo electric pipette transfers 10 nl streptavidincoated paramagnetic beads from the microliter plate into the reactionmix to separate the sequencing ladder from reaction components. Thesecond step is to flood the working area via the piezo electric pipettewith 1 μl of 0.07 M ammonium citrate to rinse the reaction mix into thehollow; this step is repeated once. The third step is to denature thesequencing ladder from the beads with 10% ammonium hydroxide solution atroom temperature. The target temperature is adjusted to RT and the piezoelectric transfers 10 nl ammonia onto the working area. The fourth stepis to cool the target again to 5° C. and redissolve the denaturedsequencing products with nl ultrapure water. In the last step, 6 nlmatrix is added into the liquid sequencing products while driving thetarget temperature again to RT to obtain optimal crystallization. Theentire SpectroChip™ microchip is transferred into a Bruker/Sequenom massspectrometer, which allows automated measurement from the nanoliterspots.

EXAMPLE 4 RNAse Digest of Ribo-modified Oligonucleotide in a Single TubeReaction

Liquid handling is performed using a modified 2 channel pipetting devicetype NP1c described in Example 1. The target site of the modifiedpipetting device has a 22W Peltier heating/cooling element controlled bya programmable thermoelectric temperature controller LFI-3526, andtemperature is measured using PT 500 type F3132 (see Example 1).

System liquid is heated to target temperature directly in the pipette.The reaction liquid is deposited on two different positions on theSpectroChip™ microchip, which contains two modified hydrophobicpositions to allow the reaction liquid to grow only in z-direction. Oneposition was used as the reaction position and the second position is adummy position to monitor the liquid loss due to evaporation (seeExample 1).

Twenty nI (50 pmol) of a ribo-modified oligonucleotide is placed on thereaction position of the 96 position SpectroChip™ microchip, while thechip temperature is maintained at 5° C. The cooled trap for the reactionliquid is used to determine the initial drop height without evaporation.From a microtiter plate, 20 nl of bovine pancreas RNase (BoehringerMannheim) is added to the reaction position. The chip then is heated toa constant temperature of 37° C. for 15 min. Following completion of thereaction, the system is cooled to 4° C. and 25 nl of matrix is added,while driving the target temperature to RT to obtain optimalcrystallization. The SpectroChip™ microchip is transferred into aBruker/Sequenom mass spectrometer for automated analysis.

The detection of degraded fragments with a mass of 4524 Daltons (Da)indicates cleavage of the oligonucleotide at the modified site.

Sequence: CGAAXTCGAGCTCGGTACCC

Ribo-modification: X is rU.

EXAMPLE 5 Exonucleolytic Degradation of Oligonucleotide in a 12 WellProcedure

Liquid handling is performed on a modified 1 channel pipetting devicetype NP1c (GeSim, Dresden Germany). As noted, this pipetting device hasa xyz table to move and dispense liquids from a piezoelectric pipette.The active piezoelectric pipette is mounted on the active block, i.e.the pipette is able to pick up samples from a microtiter plate prior todispensing reagent onto a silicon chip support (a SpectroChip™ support).97 pipettes with the same measurements as the SpectroChip™ support areconnected to the passive block. With the passive block, system liquidcan be added simultaneously to each of the 97 positions of the chip. Theactive and the passive pipettes are connected to their own pump system(diluter). Each piezo electric pipette of the active/passive block isaddressable with its own piezo electric dispensing parameters in orderto do pipette selection. The other ends of the diluters are connected toa reservoir which contains system liquid (50 mL ultrapure water). Valvesettings in the diluters allow to bypass system liquid to the piezoelectric pipette(s). The target site of the modified pipetting devicehas a 22W Peltier heating/cooling element which is controlled by aprogrammable thermoelectric temperature controller LFI-3526, andtemperature is measured via PT 500 type F3132.

System liquid is heated to target temperature directly in the pipettesof the passive block to keep the temperature gradient between reactionliquid on the target and subsequently added system liquid zero. TheSpectroChip™ microchip includes 97 modified hydrophobic positions, whichallow the reaction liquid to grow only in z-direction; 96 positions canbe used as reaction positions and the 97th position is a dummy positionto monitor the liquid loss due to evaporation (see Example 1).

Using the active piezo electric pipette, 20 nl (50 pmol) of anoligonucleotide is placed on 12 positions of the 97 SpectroChip™microchip, while the chip temperature is kept at 5° C., then 20 nl ofsnake venom phosphodiesterase (Boehringer Mannheim) is added to the 12positions. The cooled trap for the reaction liquid is used to determinethe initial drop high without evaporation. The SpectroChip™ microchipthen is heated to a constant temperature of 37° C. In order to obtain akinetic picture of the degradation reaction, the patches are allowed todry sequentially two minutes after another. The drying process ismonitored and controlled using a capacitor measurement system; systemliquid replacement utilizes the passive piezo electric pipettes.

After all reactions are completed, the chip is cooled to 5° C. and theanalyte is redissolved using the passive piezo electric pipette block.Matrix is added to the 12 positions, while driving the targettemperature to RT to obtain the best crystallization results. TheSpectroChip™ microchip then is transferred into a Bruker/Sequenom massspectrometer which allows automated measurement from nanoliter spots,resulting in twelve spectra representing the whole oligonucleotidesequence.

EXAMPLE 6 Restriction Digest of PCR Products

Liquid handling is performed on the modified 1 channel NP1c dispensingdevice described in Example 5. The target site of the modifiedNANO-PLOTTER pipetting device has a 22W peltier heating/cooling elementcontrolled by a programmable thermoelectric temperature controllerLF13526. Temperature is measured via PT 500 type F31 32 (see Example 5).

System liquid was also heated to target temperature directly in thepipettes of the passive block to keep the temperature gradient betweenreaction liquid on the target and subsequently added system liquid zero.The SpectroChip™ microchip is as described in Example 5.

A portion of exon 4 of the human apolipoprotein-E gene is amplified byPCR in a conventional 96 well microtiter plate (see Little et al., Int.J. Mass Spectrom. Ion Processes 169/170:323-330 (1997)). Aliquots ofeach well (30 nl) are transferred to a 97 SpectroChip™ microchip withthe active piezo electric pipette block while the chip temperature ismaintained at 5° C., then 20 nl of Cfol and Rsal (Boehringer Mannheim)are added to each position. The cooled trap for the reaction liquid isused to determine the initial drop high without evaporation. The chip isheated to a constant temperature of 37° C. for 15 min, during which timethe reaction volume is kept constant. After the reaction, the system iscooled to 4° C. and 25 nl of matrix is added to each position, whiledriving the target temperature to RT for optimal crystallization. Theentire SpectroChip™ microchip is transferred into a Bruker/Sequenom massspectrometer for automated analysis of the nanoliter spots.

With respect to the genotype of the genomic DNA used as template in thePCR reactions, different fragment pattern are observed. The genotypeepsilon 3 results, for example, in fragments having molecular masses of6749 Da, 7521 Da, 14858 Da, 18839 Da, 29708 Da and 33331 Da.

EXAMPLE 7 Nanoliter Liquid handling System for Real Time DNA Sequencingby Detection of Pyrophosphate Release

Sequencing by synthesis (pyrophosphate sequencing) is performed bydetecting DNA polymerase activity by an enzymatic luminometric assay,which determines the amount of inorganic pyrophosphate (“ELIDA assay”;see Ronaghi etal., Anal. Biochem. 267:65-71 (1999); Ronaghi et al.,Biotechniques 25:876-878, 880-882, and 884 (1998); Ronaghi et al.,Science 281:363-365 (1998); Ronaghi et al., Anal. Biochem. 242:84-89(1996); Nyren, Anal. Biochem. 167:235-238 (1987)).

Pyrophosphate sequencing is performed as follows. Either a synthetictemplate or a PCR product is immobilized onto a solid support.Immobilization can be performed employing standard procedures such asthe streptavidin biotin system or SIAB chemistry, as disclosed herein.In case of PCR product sequencing, the double stranded PCR product isdenatured, for example, by alkaline treatment, prior to the sequencingreaction.

An oligonucleotide primer is annealed to the immobilized templatestrand. A DNA polymerase and one deoxynucleotide triphosphate in abuffered system are added and incorporation of the nucleotide ismonitored by the release of inorganic pyrophosphate. The addednucleotide is incorporated by the polymerase only if it is complementaryto the corresponding position in the template sequence. The release ofpyrophosphate, a result of the incorporation of the nucleotidetriphosphate, is monitored as follows: inorganic pyrophosphate isconverted to ATP via the ATP sulfurylase, and the level of present ATPis monitored by the firefly luciferase system.

In the sequencing process, the addition of nucleotide triphosphates isperformed in a stepwise manner. The reaction is allowed to proceed for acertain time, then the reaction mix is separated from the solid supportand, therefore, from the immobilized template primer system, and furtherwashing is performed. Following washing, the next nucleotidetriphosphate is added as part of a reaction mix, containing allnecessary enzymes and buffers and monitoring is performed as above.Repeating the additions with all four possible nucleotide triphosphatesin a cycled manner allows stepwise (base for base) sequencing of thetemplate strand.

Liquid handling is performed on a modified 8 channel pipetting devicetype NP1c from (GeSim, Dresden Germany). As noted above, this pipettingdevice includes an xyz table to move and dispense liquids from a piezoelectric pipette. The active piezo electric pipettes are mounted on theactive block, i.e. the pipettes are able to pick up samples from amicrotiter plate prior to dispensing reagent onto a support, such as amicrochip type support SpectroChip™ microchip. 97 pipettes with the samemeasurements as the SpectroChip™ microchip are connected to the passiveblock. With the passive block system liquid can be added simultaneousonto each of the 97 positions of the chip.

The active and the passive pipettes are connected to their own pumpsystem (diluter). Each piezo electric pipette of the active/passiveblock is addressable with its own piezo electric dispensing parametersin order to do pipette selection. The other ends of the diluters areconnected to a reservoir which contains system liquid (50 ml ultrapurewater). Valve settings in the diluters allow to bypass system liquid tothe piezo electric pipette(s).

The target site of the modified pipetting device has a 22W Peltierheating/cooling element which is controlled by a programmablethermoelectric temperature controller LFI-3526. Temperature is measuredvia PT 500 type F3132. System liquid is heated to target temperaturedirectly in the pipettes of the passive block to keep the temperaturegradient between reaction liquid on the target and subsequently addedsystem liquid zero. The SpectroChip™ microchip has 97 modifiedhydrophobic positions, which allow the reaction liquid to grow only inthe z-direction; 96 positions can be used as reaction positions, one ofwhich is used to monitor the reaction using, for example, a fiber opticset very close to the reaction position. The fiber optic is connected toa photomultiplier to convert the luminescence signal to an electricsignal. A bandpass filter is placed between fiber optic andphotomultiplier. The 97th position (dummy position) is used to monitorthe liquid loss due to evaporation as described in Example 1. The wholesystem is covered and darkened to reduce background effect whileobtaining the fluorescence signal.

The universal reaction mix contains 0.1 M Tris-acetate (pH 7.75), 2 mMEDTA, 10 mM magnesium acetate, 0.1% BSA, 1 mM dithiothreitol, 5 μMadenosine 5′-phosphosulfate (APS), 0.4 mg/ml polyvinylpyrrolidone, 100μg/ml D-luciferin (bioOrbit, Finland), 4 μg/ml L-Luciferin (BioOrbit,Finland), 0.3 U/ml ATP sulfurylase (ATP:sulfate adenylyltransferase; EC2.7.7.4; Sigma Chemical Co., St. Louis, Mo.), purified luciferase(Sigma) to yield a useable luminometric response, and 2.5 U DNApolymerase (Sequenase 2.0, Amersham). Nucleotide triphosphate mixesinclude 4 separate mixes containing an aqueous solution of either 50 μMS-dATP, dCTP, dGTP and dTTP. Washing solution I is 10 mM Tris-HCl (pH7.5), 0.25 M NaCl, 0.1% Tween 20. Washing solution II is 10 mMTris-acetate (pH 7.5). Primer solution is an aqueous solution of 200 μMof sequencing primer.

PCR product is amplified from target DNA using a 5′-thiolated primer,amplification products are stored in a MTP and supplied to thenanoliquid handling device. PCR product is pipetted from the MTP on thechip using the piezoelectric pipette of the nanoliquid handling system.PCR product is immobilized on the chip surface via SIAB chemistry, andis denatured upon addition of 5 nl of 100 mM NaOH using thepiezoelectric pipette of the nanoliquid handling system. Remainingimmobilized single stranded PCR product is washed with 10 mM Tris-HCl(pH 8.0) twice.

Sequencing reactions are performed using an 8-channel piezoelectricpipette for automated performance of the cycled stepwise nucleotideaddition. One pipette is filed with water, one with the reaction mix,four pipettes are necessary to contain the separate nucleotides and twopipettes contain the washing solutions. Sequencing is performed asfollows:

1. The piezoelectric pipette of the nanoliquid handling system fillswith the sequencing primer solution of the MTP and adds it to theimmobilized template strand on the chip.

2. All 8 pipettes of the head then are filled with the respectivesolutions from the supply MTP.

3. The chip well is heated to 95° C. to denature secondary structure ofthe PCR product and is slowly cooled to RT to allow annealing of thesequencing primer. During this thermal step, evaporation is prevented bysequential addition of water droplets from a piezoelectric pipette Nr.1.

4. The primer template hybrid is washed by addition of the washingsolution I from a piezoelectric pipette Nr. 2.

5. The reaction mix is added to the primer template system using thecorresponding piezoelectric pipette Nr. 3.

6. Upon addition of the first nucleotide (SdATP) using piezoelectricpipette Nr. 4, the incorporation reaction is initiated and luminescenceis monitored; the reaction is allowed to proceed for 10 seconds, duringwhich time the reaction volume is maintained by sequential addition ofwater through pipette 1. Incorporation is identified by increasedluminescence. Intensities are analyzed to determine the number ofsequential incorporation of the respective nucleotide.

7. The reaction is stopped by rinsing the immobilized DNA with washingsolution I from pipette 2.

8. The immobilized DNA is washed by addition of washing solution II frompipette 8.

9. Reaction mix is again deposited onto the chip by pipette 3.

10. Upon addition of the second nucleotide (dCTP) using piezoelectricpipette Nr. 5, the incorporation reaction is started and theluminescence is monitored; the reaction is allowed to proceed for 10seconds, during which time the reaction volume is kept constant bysequential addition of water through pipette 1. The incorporation isidentified by increased luminescence. Intensities are analyzed todetermine the number of sequential incorporation of the respectivenucleotide.

11. The reaction is stopped by rinsing the immobilized DNA with washingsolution I from pipette 2.

12. The immobilized DNA is washed by addition of washing solution IIfrom pipette 8.

13. Reaction mix is again deposited onto the chip by pipette 3.

14. Upon addition of the third nucleotide (dGTP) using piezoelectricpipette Nr. 6, the incorporation reaction is started and theluminescence is monitored; the reaction is allowed to proceed for 10seconds, during which time the reaction volume is kept constant bysequential addition of water through pipette 1. The incorporation isidentified by increased luminescence. Intensities are analyzed todetermine the number of sequential incorporation of the respectivenucleotide.

15. The reaction is stopped by rinsing the immobilized DNA with washingsolution I from pipette 2.

16. The immobilized DNA is washed by addition of washing solution IIfrom pipette 8.

17. Reaction mix is again deposited onto the chip by pipette 3.

18. Upon addition of the fourth nucleotide (dTTP) using piezoelectricpipette Nr. 7, the incorporation reaction is started and theluminescence is monitored; the reaction is allowed to proceed for 10seconds, during which time reaction volume is kept constant bysequential addition of water through pipette 1. The incorporation isidentified by increased luminescence. Intensities are analyzed todetermine the number of sequential incorporation of the respectivenucleotide.

19. The reaction is stopped by rinsing the immobilized DNA with washingsolution I from pipette 2.

20. The immobilized DNA is washed by addition of washing solution IIfrom pipette 8.

21. Reaction mix is again deposited onto the chip by pipette 3.

22. The reaction scheme proceeds in a cycled manner by looping back tostep 6.

While the invention has been described with some specificity,modifications apparent to those with ordinary skill in the art may bemade without departing from the scope of the invention. Since suchmodifications will be apparent to those of skill in the art, it isintended that this invention be limited only by the scope of theappended claims.

8 1 25 DNA Artificial Sequence Description of Artificial SequenceSynthesized primer 1 tcacccacac tgtgcccatc tacga 25 2 25 DNA ArtificialSequence Description of Artificial Sequence Synthetic primer 2cagcggaacc gctcattgcc aatgg 25 3 25 DNA Artificial Sequence Descriptionof Artificial Sequence Synthetic probe 3 atgccccccc catgccatcc tgcgt 254 23 DNA Artificial Sequence Description of Artificial SequenceSynthetic primer 4 gactgactac ctcatgaaga tcc 23 5 20 DNA ArtificialSequence Description of Artificial Sequence Synthetic primer 5gaagctgtag ccgcgctcgg 20 6 18 DNA Artificial Sequence Description ofArtificial Sequence Synthetic primer 6 ctgcttgcca caggtctc 18 7 18 DNAArtificial Sequence Description of Artificial Sequence Synthetic primer7 cacagcaggc cagtgtgc 18 8 16 DNA Artificial Sequence Description ofArtificial Sequence Synthetic primer 8 gaggcccatc ctcacc 16

What is claimed is:
 1. A system for performing a reaction, comprising: asupport for performing the reaction; a nanoliter dispensing pipette fordispensing a submicroliter amount of a liquid onto the support; atemperature controlling device for regulating the temperature of thesupport; and means for controlling the amount of liquid dispensed,wherein: the amount of liquid dispensed corresponds to the amount ofliquid that evaporates from the support; and the controlling meanscomprises software that calculates the amount of liquid that evaporatesand signals the dispensing pipette to deliver an amount of liquid thatcorresponds to the amount that evaporates, wherein the system is open.2. The open system of claim 1, further comprising a means fordetermining the temperature of a liquid on the support.
 3. The opensystem of claim 1, wherein the support comprises a bead, pin, comb,wafer, well or microchip.
 4. The open system of claim 1, wherein thesupport is capable of linking to a biopolymer or to a biologicalparticle.
 5. The open system of claim 1, further comprising a detectionsystem for monitoring the reaction.
 6. The system of claim 1, whereinthe support comprises a surface for containing a submicroliter reactionvolume that comprises an array of hydrophilic regions and adjacenthydrophobic regions.
 7. The system of claim 1, further comprising asupport having a target site for retaining or containing a reactionmixture.
 8. A system, comprising: a solid support having a target sitefor retaining or containing submicroliter quantities of a liquid; aliquid dispensing system for dispensing a liquid to the target site; atemperature controlling system, which regulates the temperature of thesolid support; and an interface for indicating an amount of liquid to bedispensed to the target site, wherein the amount of liquid to bedispensed corresponds to an amount of liquid that evaporates from thetarget site.
 9. The system of claim 8, wherein the amount of liquid tobe dispensed is determined based on the temperature of the liquid on thesolid support.
 10. The system of claim 8, wherein the interface monitorsthe level of a liquid on the target site.
 11. The system of claim 10,wherein the interface comprises an electrical circuit comprising theliquid on the target site.
 12. The system of claim 8, wherein thetemperature controlling system comprises a Peltier element.
 13. Thesystem of claim 8, further comprising a detection system.
 14. A system,comprising: a solid support having a target site, which can contain asubmicroliter amount of a liquid; a liquid dispensing system, which candispense a liquid to the target site; a temperature controlling system,which regulates the temperature of the solid support; and means forregulating an amount of liquid dispensed from the liquid dispensingsystem, wherein the amount of liquid dispensed corresponds to an amountof liquid that evaporates from the target site, wherein the system isopen.
 15. The system of claim 14, wherein the regulating means comprisesan interface in communication with the target site.
 16. The system ofclaim 14, wherein the regulating means comprises manual input.